Notebook

Interlab

The purpose of this year’s interlab is to address the high level of variability in fluorescence measurements. Typically, fluorescence is measured using the optical density (OD), or the absorbance of light by a sample when measured at 600nm using a plate reader, nanodrop, or spectrophotometer. However, since this method can be inaccurate, Interlab seeks to improve the standard method of measuring fluorescence in populations of cells. Since the number of cells in a sample can vary, if the fluorescence is normalized to the cell count of the sample, then the average amount of fluorescence can be calculated, which is likely more accurate than that of a population of unknown size. Therefore, Interlab will investigate if normalizing fluorescence measurements to colony-forming units (CFUs) will yield more precise results than OD.

Each iGEM team was asked to normalize the absorbance to the number of cells in two ways: by comparing sample absorbance to absorbance of a sample of known concentration of beads similar in size to E. coli cells and by counting CFUs from each sample.

OD

For the first method, absorbance was measured for a sample and serial dilution of silicon beads. These beads, similar in size and shape to E. coli cells, are expected to scatter light in a similar manner, providing an analogous sample of known concentration. The first part of this process was to get a reference point for OD₆₀₀ to easily interchange between Abs₆₀₀ and OD₆₀₀, accounting for differences in volume between wells. In this measurement, four equal samples LUDOX CL-X (a 45% colloidal silica suspension) were compared to samples of ddH₂O, and the OD was measured using a plate reader. By finding the mean of the replicates, correcting by subtracting the mean of the water to get just the absorbance of the silica, and dividing by the OD₆₀₀ from the provided Excel sheet, the conversion factor (OD₆₀₀/Abs₆₀₀) was calculated to be 3.500.

A particle standard curve was also developed using a serial dilution of monodisperse silica microspheres to plot the concentration versus absorbance, thus allowing the estimation of the number of cells based off the absorbance. The serial dilution was performed four times, and the Abs₆₀₀ was measured to create the following standard curves:

Finally, a fluorescence standard curve was created to have a point of comparison between outputs of different plate readers, getting rid of the arbitrary units assigned by different instruments. Fluorescein is used as an easier, lower-cost alternative to GFP as it has similar excitation and emission properties. Four identical serial dilutions were completed for fluorescein, and the Abs₆₀₀ was measured to create the following standard curves:

CFU

This part of the Interlab found the volume of a 0.1 OD₆₀₀ culture and the number of cells in a known volume by growing a sample and counting the number of colonies that grew. For the “Starting Sample Preparation,” the OD₆₀₀ of diluted overnight cultures of positive and negative controls were taken along with that of blank media. Based on the starting sample, overnights were diluted in triplicate, and the OD₆₀₀ was taken and adjusted using the blank, yielding the absorbance. To find the number of cells in a given volume, a serial dilution was performed for each sample, and the last three dilutions were plated and grown overnight. Colonies were counted, and, with the understanding that one cell grows into one colony, the number of cells per unit of volume was found. To standardize across dilutions, we used the equation:

number of colonies x final dilution factor = CFU/mL

Dilution 3

Plate	Number of Colonies	CFU/mL 0.1 OD₆₀₀ solution
1.1	185	14800000
1.2	28	2240000
1.3	79	6320000
2.1	Over 300	N/A
2.2	Over 300	N/A
2.3	Over 300	N/A
3.1	121	9680000
3.2	261	20880000
3.3	170	13600000
4.1	94	7520000
4.2	270	21600000
4.3	Over 300	N/A

Dilution 4

Plate	Number of Colonies	CFU/mL 0.1 OD₆₀₀ solution
1.1	18	14400000
1.2	7	5600000
1.3	6	4800000
2.1	65	52000000
2.2	105	84000000
2.3	76	60800000
3.1	42	33600000
3.2	26	20800000
3.3	12	9600000
4.1	31	24800000
4.2	24	19200000
4.3	33	26400000

Dilution 5

Plate	Number of Colonies	CFU/mL 0.1 OD₆₀₀ solution
1.1	1	8000000
1.2	0	0
1.3	1	8000000
2.1	1	8000000
2.2	2	16000000
2.3	8	64000000
3.1	1	8000000
3.2	3	24000000
3.3	1	8000000
4.1	0	0
4.2	5	40000000
4.3	3	24000000

Lab Notebook

Click on each section to view the week's notebook.

May

May 22, 2018

Competent Cell Prep

Liz, Peter, Ryan, Shannon, Sricharan

We’re preparing competent cells (E. coli, XL1 Blue) for the summer.
Craig and Sricharan tried to do this in December, but it didn’t work since the shaker was at 200rpm instead of at 250rpm.

Today: prepping stock solutions, streaking plate

TfbII (50 mL)

Reagent	Concentration	Amount needed	Amount measured
MOPS	10mM	0.104g	0.1040g
CaCl₂	75mM	0.417g	0.4160g
RbCl₂	10mM	0.061g	0.0616g
Glycerol	15% (v/v)	7.5mL	7.5mL

Measured amounts of reagents in flask (next time, use beaker)
Added most of water (don’t top off, so you can adjust pH)
pH (w/stirring if possible) to 6.5, using 1M KOH dropwise - got to 6.52… (make sure to standardize with pH 7 and 4 buffers)
Topped off in graduated cylinder
0.45µm filter sterilized
No need to autoclave this solution
Stored in Tigger (4°C)

TfbI (200 mL)

Reagent	Concentration	Amount needed	Amount measured
Potassium Acetate	30mM	0.588g	0.589g
MnCl₂	50mM	1.98g	1.99g
CaCl₂	10mM	0.222g	0.224g
RbCl₂	10mM	0.242g	0.242g
Glycerol	15% (v/v)	30mL	30mL

Measured amounts of reagents in flask (next time, use beaker)
Added most of water (don’t top off, so you can adjust pH)
pH (w/stirring if possible) to 5.8, using .2M acetic acid dropwise - got to 5.82… (make sure to standardize with pH 7 and 4 buffers)
Topped off in graduated cylinder
0.45µm filter sterilized
No need to autoclave this solution
Stored in Tigger (4°C)

ᴪa (500 mL)

Reagent	Concentration	Amount needed
Bacto yeast extract	5g/L	2.5g
Bacco tryptone	20g/L	10g
MgSO₄	5g/L	2.5g
KOH	1M	Added dropwise until solution reached pH 7.6

Dry substances mixed in bottle (would be easier to use a beaker)
Water topped off to almost 500mL
Pour into flask to adjust pH using spinner

Standardized pH meter using 7 and 10 buffers, pain to do

Pour back into graduated cylinder to top off to 500 mL
Use sterile filter
Aliquot 100mL into two 300mL flasks with caps, remaining 300mL in bottle
Autoclave at liquid 30, 45 minutes dry
Removed from autoclave, allowed to cool, and transferred to 4℃ fridge (Tigger)

Also streaked Ψa plate

Sterile environment (with portable burner nearby)
Stabbed competent XL1 Blue cells (frozen stock) with a pipet tip
Gently spread frozen piece of cells onto plate with tip (be careful not to puncture agar)
Spread cells on plate with Pasteur spinner (not the plating beads, @Craig)
Put plate into incubator at 39°C (not 37°C, ugh)

May 22, 2018

Interlab Calibration

Liz, Peter, Ryan, Shannon, Sricharan

Calibration 1, OD₆₀₀ Reference Point - LUDOX protocol:

100µL LUDOX CL-X in wells A1, B1, C1, D1
100µL ddH₂O in wells A2, B2, C2, D2

Absorbances

	1	2
A	0.045	0.024
B	0.041	0.025
C	0.042	0.025
D	0.042	0.025

Wavelength: 600nm
Optics: top
Temperature: 25.9°C

Calibration 2

Stock Solution (Vortex silica beads for 30 sec. before adding):

Reagent	Amount
Silica beads	96 µL
ddH2O	904 µL

Add 100 µL ddH₂O to wells A1, B1, C1, D1, …, A12, B12, C12, D12
Add 200 µL stock solution to A1, B1, C1, and D1 (vortex 10s before adding)
Transfer 100 µL from A1 to A2, pipet to mix, transfer 100 µL from A2 to A3, pipet to mix, etc. until A11. Repeat for B, C, and D.

Cells A1, B1, C1, and D1 should be pure stock
Cells A12, B12, C12, and D12 should be pure ddH₂O

Absorbance

	1	2	3	4	5	6	7	8	9	10	11	12
A	1.075	0.545	0.343	0.152	0.087	0.055	0.051	0.032	0.028	0.031	0.028	0.025
B	1.199	0.666	0.306	0.161	0.086	0.057	0.04	0.033	0.028	0.026	0.026	0.025
C	1.191	0.651	0.346	0.176	0.091	0.03	0.051	0.043	0.031	0.028	0.027	0.025
D	1.325	0.587	0.299	0.168	0.088	0.057	0.039	0.032	0.027	0.026	0.025	0.025

Wavelength: 600nm
Optics: top
Temperature: 26°C

Calibration 3: Fluorescence Standard Curve

Fluorescein stock solution:

Reagent	Amount
Fluorescein	Provided in Kit (5 tubes)
1x PBS pH 7.4-7.6	10 mL

Spun down fluorescein in tube so the pellet was at the bottom
Made ~ 40 mL 1x PBS in falcon tube (originally used 10 x accidentally for first 3 attempts/ tubes)
Used 1 mL of 1x PBS to resuspend fluorescein in kit tube > makes 10x fluoresceiµLtocol calls for pH of 7.4-7.5. Problem? > email iGEM
Diluted 10 fluorescein stock with 900 µL of 1x PBS and 100 µL of 10x fluorescein stock to make 1x fluorescein solution (10 M)

Serial Dilutions:

Used clear flat-bottom 96 well plates
Put 200 µL of 1x fluorescein into column 1 rows A, B, C, D
Put 100 µL of 1x PBS in columns 2-12 for rows A, B, C, D

Column 12 for all rows (A, B, C, D) is control and only contains 100 µL 1x PBS

Transferred 100 µL of fluorescein stock from A1 to A2
Mixed A2 > pipetting up and down 3 times
Transferred 100 µL from A2 to A3 and mixed A3
Repeated until after mixing A11 > then 100 µL removed from A11 and put into liquid waste
Process described for row A repeated for all other rows (B, C, D)

Fluorescence

	1	2	3	4	5	6	7	8	9	10	11	12
A	497	266	144	75	41	23	14	9	8	6	6	5
B	532	302	153	81	44	24	25	10	7	7	5	5
C	532	295	154	81	45	25	15	10	7	7	6	5
D	547	297	159	83	45	25	16	11	8	7	6	5

Excitation: 485 nm /20 nm
Emission: 528 nm /20 nm
Optics: top
Temperature: 25.8°C (room temp)

May 23, 2018

Competent Cell Prep

Sricharan

Took the Ψa plate out around 9am

It was densely packed with colonies (tan color), but some isolated ones appeared towards one edge of the plate

Kept plate at 4°C cold room during day
Overnight cultures prepared near portable burner (to the delight of Toni watching me try and refill the burner)

Picked 3 colonies with P200 tip
Each colony into a separate round bottomed tube with loose caps, filled with Ψb media
Tubes placed in shaker at 37°C and 250rpm around 4:30pm

May 24, 2018

Competent Cell Prep

Liz, Peter, Shannon, Sricharan

Took the overnight cultures out around 9:15am

They all are cloudy compared to the original media, so decent growth it seems
Will start subcultures around noon
Cultured in shaker for an hour and 22 minutes and we over grew both cultures by a little
Prepared another overnight culture

May 24, 2018

Competent Cell Prep

Liz, Peter, Shannon, Sricharan

Took the overnight cultures out around 9:00am

Growth in both cultures
Pre-warmed Ψb media before subculturing
Subcultured in Ψb media for 45 minutes (Flask A around 0.36 OD600; Flask B around 0.38 OD600)
Checked again at 55, then 58 minutes and each was around 0.48 OD600
Spun cells in pots in MSE 18 to isolate pellet and removed supernatant
Resuspended in TfbI and spun again and removed supernatant
Resuspended in TfbII, pooled A and B, and chilled on ice for 15 minutes
Aliquoted 200 µL of cell solution into autoclaved microcentrifuge tubes and snap-froze using liquid nitrogen
Put competent cells into -80 fridge on second floor

May 29, 2018

Interlab Transformation

Shannon, Liz

Plasmid Re-suspension

Add 10 µL dH2O to wells 2D, 2B, 2F, 2H, 2J, 2L, 2N, and 2P on plate 7 from the interlab kit and pipette to resuspend the DNA in each well. Let sit for a few minutes. Transfer to 1.5 mL tubes.

Note: 2D and 2B were accidently added to the same 1.5 mL tube, so these two were re-done using 21B and 21D from kit plate 7 from the 2017 interlab kit, which have the same corresponding DNA.

May 29, 2018

Chloramphenicol Plate Preparation

Ryan, Peter, Sricharan

Preparing Chloramphenicol Stock

Made 10 mL stock of 35 mg/mL chloramphenicol in EtOH (this is 1000x as specified by iGEM)
Took from Rachel’s floor (we bought it though), 0.3496g made up to 10mL volume

Preparing Chloramphenicol LB Plates (24)

Collected ingredients for LB media and added to beaker
Q/s’d with dH₂0 to 400
Mixed in beaker for 20 min and Q/s’d to about 450 mL with spray bottle to get solid particles off of the sides of the beaker and into the solution
Tried pouring into grad cylinder to finish Q/s but still goupy
Poured ~ 50 mL of dH20 into grad. Cylinder to capture the goop stuck to the bottom after pouring as much as we could back into the beaker. Poured this 50 mL back into the beaker as well
Turned up temp and stir speed on hot plate and stirred for another 25 minute in beaker
Poured into bottle and stirred (stopping frequently to shake bottle) for ~ 30 minutes
Autoclaved on S03 setting for 30 minutes (autoclaving less than a liter of liquid)
Added 0.5 mL of chloramphenicol (1000x, 35 mg/mL) to solution
Plated LB + chloramphenicol onto 24 plates and put in cold room (-4 degrees C)

Rushed putting the plates away, so there might be some condensation on plates

May 30, 2018

Interlab Transformation

Shannon, Liz, Ryan

Thawed DH5-alpha competent cells on ice
Pipet 50 μL competent cells into empty 1.5 mL tubes
Pipet 1 μL resuspended DNA (from yesterday, see page above) into individual tubes (according to well number) with competent cells

DNA from tubes: 21B, 21D, 2F, 2H, 2J, 2L, 2N, and 2P in box 1 from freezer

Pipet 1 μL control DNA (10pg/μL) into control tube with competent cells
Incubate tubes on ice for 30 min (0:00-30:23)
Heat shock at 42℃ for 45s (30:23-31:08)
Pipet 950 μL SOC media at room temperature into each tube
Incubate at 37℃ for 1 hr at 300 rpm
Plate 100 μL transformation (lower concentration)

Condensation got on agar, tried tapping plates to remove water but some remained
Spread water with spinners before putting transformation on plates
Unsure if plates N and P got switched

Spin down cells at 6800g for 3 min and discard 800 μL supernatant
Resuspend pellet in the remaining 100 μL and plate (higher concentration)

Amount of liquid remaining for resuspension varied from tube to tube

Incubate at 37℃ for 14-18 hours

Put into incubator ~6pm
Incubator is kept at 39℃
Checked after 15.5 hours, barely any growth

May 30, 2018

Competent Cell Transformation (Scott-Simanis, Test Kit)

Peter, Sricharan

2 vials of Scott-Simanis competent cells from 5/25/18 thawed at RT (200 μL in each tube)
After thawed, placed on ice for 10 minutes

At 8 minutes, aliquoted 50 μL (150 μL from each tube) into 6 separate tubes
At 10 minutes, added 1 μL of DNA from Competent Cell Test Kit

Duplicates of 10, 50, 100 pg/μL BBa_J04450, RFP construct

Chilled aliquots on ice 30 min
Heat shocked aliquots at 42 °C for 90 s
Incubated on ice for 90 s
Added 200μL Ψb media to each aliquot
Incubated at 37 °C, 300rpm for 1 hour
Plated onto LB plates with chloramphenicol (35 μg/mL) with spinners, under portable burner
Incubated at 39 °C incubator downstairs starting at 6pm

May 31, 2018

LB + Chloramphenicol Media Preparation (w/o agar)

Peter, Sricharan, Liz, Shannon, Ryan

Used for inoculation for O.C. & dilutions + control for transformation fluorescence readings

Preparing Chloramphenicol LB media (500 mL)

Collected and measured out ingredients for LB media and added to bottle
Q/s’d with dH₂0 to 400 mL and shook bottle to dissolve ingredients
Stirred in beaker for 15 min
Poured into grad cylinder to finish Q/s-ing to 500 mL
Poured back into bottle and stirred for ~ a minute
Autoclaved on S03 setting for 30 minutes (autoclaving less than a liter of liquid)
Added 0.5 mL of chloramphenicol (1000x, 35 mg/mL) to solution

May 31, 2018

Interlab Overnight Cultures

Peter, Sricharan, Liz, Shannon, Ryan

Condensation on plates = osmotic stress on cells…. Not a lot of growth

Checked plates at 9am - not a lot of growth
Took plates out at 12pm, at least a few colonies on most plates with concentrated transformations, except Wells 21D (2017), 21B (2017)

Made 2 OCs for each well with most well defined/isolated colonies from each well

Put into “new” old shaker in classroom (cleaned in morning with 70% ethanol)
220rpm, 37 °C

June 1, 2018

Interlab Fluorescence Measurements

Peter, Sricharan, Liz, Shannon, Ryan, Toni

Grabbed OCs @ 9:15am from shaker
Diluted 0.5mL of OCs 10x, put foil on diluted tubes
Took Abs₆₀₀ readings on plate reader (100μL samples)

Samples on Plate	1	2	3	4	5	6	7
G	21D (‘17)	2F-1	2H-1	2J-1	2L-1	2N-1	2P-1
H		2F-2	2H-2	2J-2	2L-2	2N-2	2P-2

Since Abs=ebc, and we need to dilute to a target Abs₆₀₀ of 0.02, and final volume of 12mL, Valiquot=0.24/Abs₆₀₀mL. Also, V_media = 12mL-V_aliquot

Need to put in values from data on computer

Abs₆₀₀	1	2	3	4	5	6	7
G
H

V_aliquot(mL)	1	2	3	4	5	6	7
G	7.7	4.6	2.3	2.3	1.7	2.8	2.1
H		1.8	2.2	2.3	2.0	3.1	2.4

V_aliquot(mL)	1	2	3	4	5	6	7
G	4.3	7.4	9.7	9.7	10.3	9.2	9.9
H		10.2	9.8	9.7	10.0	8.9	9.6

@12pm Put diluted & absorbance-corrected cultures back in shaker

220rpm, 37 °C for 6 hours
Used 0.5mL aliquot of these cultures for t = 0h readings

Placed on 96-well plate as in protocol
Note we don’t have negative or positive controls… Need to redo next week

June 1, 2018

LB + Chloramphenicol Plates Preparation

Peter, Ryan

To be used for competent SS cell test & positive and negative controls for Interlab

Preparing Chloramphenicol LB plates (24)

Collected and measured out ingredients for LB media and added to bottle
Q/s’d with dH₂0 to 400 and shook bottle to dissolve ingredients
Stirred in beaker for 30 min
Poured into grad cylinder to finish Q/s to 500 mL
Poured back into bottle and stirred for ~ a minute
Autoclaved on S03 setting for 30 minutes (autoclaving less than a liter of liquid)
Added 0.5 mL of chloramphenicol (1000x, 35 mg/mL) to solution
Let plates cool for an hour on lab bench
Put them in the hood with the plates half open w/ agar side on the bottom to get rid of condensation (should have done this immediately to dry them)

June 1, 2018

Title

Names

June 1, 2018

Title

Sricharan, Liz

	3	4	5	6	7	8	9
A (absorbance)
A (fluorescence)
B (absorbance)
B (fluorescence)
C (absorbance)
C (fluorescence)
D (absorbance)
D (fluorescence)
E (absorbance)
E
F(absorbance)
F (fluorescence)
G
G (fluorescence)
H
H (fluorescence)

June

June 4, 2018

LB + Chloramphenicol Plate Prep

Ryan

See protocol from 6/1/18 for LB + Chloramphenicol Plate Prep

Notes:

Made 23 plates (whoops)
Burned serological pipette...
… and part of a plate

June 4, 2018

Interlab Transformation of 21B and 21D

Liz

See protocol from 5/29/18 transformation

Deviations/Notes

Plates were taken out of fridge and warmed to room temperature

No condensation on plates because they were allowed to mostly cool on the bench and then transferred to the hood with the lids partially off in the process of making the plates

Only doing DNA from wells 21B and 21D (from 2017 kit plate 7)

Colonies didn’t grow on plates last time (5/29)
For each sample, made 1 dilute plate and 2 concentrated plates with the hope of actually getting colonies this time

Forgot to prechill tubes before putting in competent cells and resuspended DNA
Centrifuged at 12:45 during 30 minute incubation on ice
Only ended up making 1 concentrated plate each for 21B and 21D
Put in incubator at 4:20pm

Times

0:00-30:00 - incubate on ice for 30 minutes
30:00-30:45 - heat shock at 42℃ for 45 s
30:45-35:45 - incubate for ice for 5 min

June 4, 2018

SS Competent Cell transformation Test (Day 1)

Peter

See protocol “Competent Cell Transformation (Scott-Simanis, Test Kit)” from 5/29/18 transformation test

Deviations/Notes

Plates were taken out of fridge and warmed to room temperature

No condensation on plates because they were allowed to mostly cool on the bench and then transferred to the hood with the lids partially off in the process of making the plates

Ψb taken out of fridge to warm to room temperature
Only made samples from tube 1 for 10 pg/ μL
Took two tubes of competent cells from -80 degrees celsius
Took ~ 7 minutes to add DNA (too long for cells to be warm during this step)
Centrifuged at 12:45 during 30 minute incubation on ice (should have done this before)
Re-plated 100 pg/ μL plate 1 (labeled new plate “1b”) because burned cells with pasteur pipette for plate 1a
Potentially killed cells for 10 pg/ μL on plate 1b
Put in incubator at ~ 4:20pm

June 5, 2018

SS Competent Cell transformation Test (Day 2)

Peter

Checked cells @ 9:15 am. Some growth on 100 pg/ μL plate 1a and no visible growth on others
Checked again @ 10:15 am and same growth situation

June 5, 2018

Checking Distribution Kit Plasmids

Sricharan, Liz, Peter, Shannon

Needed to run a gel for 2017 kit 7 wells 21B and 21D to check to see if the plasmid was there because the transformation yielded no colonies again

Used 2017 kit 7 wells 21B and 21D, 2017 kit 3 well 8P, and 2017 kit 2 well 6F
Originally thought we had to do a digest of plasmids before running gel so measured concentration of DNA in samples using nanodrop (measure 1μL)

Blank with water first

Results

Kit 7 well 21B: 134.4 ng/μL
Kit 7 well 21D: 109.0 ng/μL
Kit 3 well 8P: 114.7 ng/μL
Kit 2 well 6F: 131.7 ng/μL

Made 2 small agarose gels (1%)
25mL 1x TAE, 0.25g agarose warmed in microwave
2.5 µL SYBR Safe added

All wells in gel from 2017 Kits

Well	1	2	3	4	5	6	7	8	9	10
Item	2-Log Ladder	Kit 7, Well 21D	Kit 7, Well 21B	Kit 2, Well 6F	Kit 3, Well 8D	2-Log Ladder	Test Kit 100pg/µL	Test Kit 50pg/µL	Test Kit 10pg/µL	Linearized pSB1C3

Ran 52 minutes @ 50V on mini gel rig

No condensation (new TAE)

Image on transilluminator hard to see
Similar gel image - only clear lanes were ladder and linearized backbone

Diffuse spots for plasmids in wells 2-5, we should be fine

June 6, 2018

Redoing Interlab and Scott-Siamanis Transformations

Sricharan, Liz, Peter, Shannon

Redoing transformations from earlier this week (since they didn’t work)

Using water bath at 42°C instead of heat block for shocking cells
Keeping all tubes prechilled/on ice
Prewarming SOC media
Using incubator to shake cells <300rpm instead of thermomixer

This incubator thing is probably overkill, we’ll go back to thermomixer later

Got cells (2 tubes SS, 1 tube DH5-alpha) from -80°C

SS thawed off ice ~3min
DH5-alpha thawed on ice ~5min

Immediately portioned 50µL aliquots of cells into several 1.5mL tubes

Scott-Simanis cells - samples

Competent Cell Test Kit

100pg/µL
50pg/µL
10pg/µL

Well 2F, Plate 7, 2018 Kit

DH5-alpha cells - samples

Competent Cell Test Kit, 10pg/µL
pUC19 Control
Plate 7, 2018 Kit

Well 4B (0.2 µL)
Well 4B (1 µL)
Well 4D (0.2 µL)
Well 4D (1 µL)
Well 2F (1 µL)

For SS

Incubate 10 minute on ice, add DNA (1 µL), incubate on ice 30 min

For DH5-alpha

Add DNA immediately (1 µL or 0.2 µL if indicated on sample name), incubate on ice 30 min

Heat shock @ 42°C in water bath, 45 seconds (DH5-alpha) or 90s (SS)

Accidentally dropped DH5-alpha samples into water, held in water after

Incubate on ice 5 min (DH5-alpha) or 90s (SS)
Add (prewarmed) 950µL SOC media to each DH5-alpha tube, and 200µL Ψb into each SS tube
Incubate @ 37°C in shaker in classroom ~250rpm
Plated 100µL on LB+Cam plates

For DH5-alpha, followed iGEM protocol to concentrate solutions
Only plated for DH5-alpha controls after concentrated

Incubated at 39°C downstairs ~4:30pm

June 7, 2018

Interlab Positive and Negative Controls

Sricharan, Liz, Peter, Shannon

Took plates from yesterday out ~9:30am

Good growth on DH5-alpha plates (Interlab)

Except pUC19 control, which was aliquoted after comp cell tube was dropped into ethanol

No growth on most Scott-Simanis plates (not even Well 2F)

We know cells are not competent now - plates and protocol are fine

Will do overnight cultures for Interlab cells and for new batch of competent cells this afternoon

June 7, 2018

Psi Solutions

Liz, Peter

Psi A

Substance	Concentration	Protocol Amount	Actual Amount
Bacto yeast extract	5 g/L	0.625 g	0.6357 g
Bacco tryptone	20 g/L	2.5 g	2.5090 g
MgSO₄	5 g/L	0.625 g	0.6261 g
Bacto agar	14 g/L	1.75 g	1.7512 g

Fill with dH₂O to just under 250 mL

This was our mistake!! We used enough chemicals to make 125 mL but put in too much water, making the solution useless

Add 1M KOH dropwise until pH reached 7.63
Mixed in beaker using stir plate

Didn’t mix fast enough so agar clumped on sides and at bottom
Shook a lot in a bottle and poured back and forth to get out as much agar as possible

Autoclaved on liquid setting
Was about to pour plates but realized that we can’t use because too diluted

Substance	Concentration	Protocol Amount	Actual Amount
Bacto yeast extract	5 g/L	2.5 g	2.50 g
Bacco tryptone	20 g/L	10 g	10.0002 g
MgSO₄	5 g/L	2.5 g	2.5026 g

Fill with dH₂O to just under 500 mL
Mixed in beaker using stir plate
Adjusted pH to 7.60 by adding 1M KOH dropwise
Topped off to 500 mL
Put through a sterile filter and aliquoted 100mL each into 3 flasks and the rest into a bottle
Autoclaved on liquid setting
Store in fridge (4°C)

June 7, 2018

Chloramphenicol plate prep

Shannon and Toni

See protocol from 6/1 - made 1L
Mixture was goopy when transfering from bottle to graduated cylinder to Q/s to 500mL, so had to put back in bottle and shake/continue mixing on hot plate. Rinsed graduated cylinder with dH₂O to get goop back into bottle (not enough to go past 500mL).

June 11, 2018

Interlab CFU Day 1

Sricharan, Liz, Shannon

Circled three colonies (labeled a, b, c) from plates 4B 1𝜇L and 4D 1𝜇L (made 6/6/18)
Pipetted 5 mL LB broth into OC tubes

Six tubes: 4Ba, 4Bb, 4Bc, 4Da, 4Db, 4Dc
5mL in each tube

Used 200 1𝜇L pipet tip to take colony a from plate 4B and drop into tube 4Ba
Repeat with other circeled colonies into corresponding tubes
Put into shaker at about 37℃ and 220 rpm overnight

In: 3:30 pm

June 11, 2018

SS Competent Cells Trasformation Test Day 1

Peter, Deivydas, Ryan

See protocol “Competent Cell Transformation (Scott-Simanis, Test Kit)” from 5/29/18 transformation test

Deviations/Notes

Plates were taken out of fridge and warmed to room temperature

No condensation on plates because they were allowed to mostly cool on the bench and then transferred to the hood with the lids partially off in the process of making the plates

Ψb taken out of fridge to warm to room temperature
Made one sample/ plate for each concentration (10 pg/ μL, 50 pg/ μL, 100 pg/ μL) adn well 2F from interlan study as a control
Took one tube of competent cells from -80 degrees celsius
Let ice thaw for ~ one minute
Centrifuged during 30 min. To make sure is mixed (should not have done this- cells are fragile)

June 12, 2018

SS Competent Cells Trasformation Test Day 2

Peter, Deivydas, Ryan

Little to no growth - ask Rachel what we are doing wrong
Three colonies on 100 pg/ μL

June 12, 2018

Interlab CFU Day 2

Sricharan, Liz

Took overnight cultures of positive (4B) and negative (4D) controls
Following CFU protocol

C₁V₁ = C₂V₂→ V₁ = C₂V₂/C₁ = 0.1(1000mL)/8(1:8OD₆₀₀-blank OD₆₀₀)

Wells

	1	2
F	1 (positive control)	2 (positive control)
G	3 (negative control)	4 (negative control)
H	Blank media	Blank media

OD₆₀₀

	1	2
F	0.237	0.122
G	0.250	0.208
H	0.033	0.034

Culture Volume (checked OD₆₀₀ with plate reader)

V₁: 61μL culture, 939μL media
V₂: 141μL culture, 859μL media
V₃: 58μL culture, 942μL media
V₄: 72μL culture, 928μL media

Shannon is making more LB+Cam media for our dilutions

Plate notation: A.B.C

A=culture number (1,2=positive control, 3,4=negative control)
B=sample number
C=dilution number (3, 4, and 5 are plated)

Diluted according to protocol, plated cells in dilutions 3-5

Reused 15mL Falcon tubes for dilutions 1-3

Incubate at 39℃ downstairs overnight

In at ~4:15

June 12, 2018

SS Competent Cells Trasformation Test Day 1

Peter

New Protocol - we were doing stuff wrong

Retrieved 3 vials of Scott-Simanis competent cells (XL1 Blue from 5/25/18, 6/8/18, and Rachel’s cells) from -80 and brought upstairs on ice (200 μL in each tube)
Thawed at RT for ~ one minute
NO ALIQUOTING AS PIPETTING SHREDS FRAGILE CELLS
Added 1μL of DNA immediately after thawed (BBa_J04450 100 pg/μL from 2018 CC test kit)
NO CENTRIFUGING TO MIX DNA IN
Chilled samples on ice for 30 minutes
Heat shocked samples WITH WATER BATH at 42 °C for 45 S
Incubated on ice for ~ 90 s
Added 800μL (4 times volume of samples) 2YT media to each sample
Incubated at 37 °C ON HEAT BLOCK (NO SHAKING) for 60 min
Plated out w/ flame two plates for each sample: 100 μL on plate 1 and 200 μL on plate 2
Incubated overnight @ 37 °C

June 13, 2018

SS Competent Cells Trasformation Test Day 2

Peter

Retrieved from the incubator @ 9:15 (~ 17 hours in the incubator)
FINALLY IT WORKED MOSTLY!!!

Colony Growth:

	Rachel (Date?)	iGEM 5/25/18	iGEM 6/8/18
Plate 1 (100 μL)	~ 15 colonies	1 colony??	13 colonies
Plate 2 (200 μL)	69 colonies	3 colonies	~42 colonies

June 13, 2018

Counting Interlab Colonies

Liz, Sricharan, Shannon, Peter

Dilution 3

Plate	Number of Colonies
1.1	185
1.2	28
1.3	79
2.1	Over 300
2.2	Over 300
2.3	Over 300
3.1	121
3.2	261
3.3	170
4.1	94
4.2	270
4.3	Over 300

Dilution 4

Plate	Number of Colonies
1.1	18
1.2	7
1.3	6
2.1	65
2.2	105
2.3	Over 76
3.1	42
3.2	26
3.3	12
4.1	31
4.2	24
4.3	33

Dilution 5

Plate	Number of Colonies
1.1	1
1.2	0
1.3	1
2.1	1
2.2	2
2.3	8
3.1	1
3.2	3
3.3	1
4.1	0
4.2	5
4.3	3

June 18, 2018

Transforming Parts from Registry

Sricharan

Resuspended some parts from 2018 Distribution Kit to transform tomorrow

Plate	Well	Part	Name (BBa_)
4	17D	Anderson Promoter (Strong)	J23100
4	19D	Anderson Promoter (Medium)	J23110
4	19J	Anderson Promoter (Weak)	J23114
1	17H	Ptac	K864400
5	17J	‘tesA	K1472601
5	7K	TUDelft mtrCAB	K1316012
6	21K	ADC (Petrobrick)	K590031
6	7K	AAR (Petrobrick)	K590032

Resuspending each well w/ 10μL of dH2O, let sit a few minutes, aliquoted into 1.5mL tubes.

June 19, 2018

2xYT Medium

Liz

Wanted to make 500mL for use for transformations/potentially overnight cultures (point is we don’t need a ton)

In a bottle measured:

450mL dH₂O
8g tryptone (measured 8.00g)
5g yeast extract (measured 5.00)
2.5g NaCl (measured 2.50)

Shook bottle between adding each substance to dissolve

When dissolve adjusted pH to 7.0 with 5M NaOH

Added dH₂O up to ~480mL before adjusting pH
Only had 1M NaOH
Had to use pH 4 calibration solution because start out acidic

Top off to 500mL

Autoclave for 20 min at 15 psi (1.05kg/cm³) on liquid cycle

Used 30 minute liquid setting

Put in cold room to cool before using for transformations

June 19, 2018

Making CAM and Amp plates

Peter

See protocol from 6/1/18

Notes/Deviations

After autoclaving, split 500 mL into two flasks of 250 mL each
Added 250 μL of chloramphenicol (35 mg/ mL stock) to one flask
Added 250 μL of Ampicillin (35 mg/ mL stock) to other flask

June 19, 2018

SS Competent Cells Trasformation Test Day 1

Peter, Shannon, Charan

Measured 0.5043g ampicillin (Na salt) from Rachel’s lab to make 100mg/mL stock solution (1000x) in 50% EtOH

See protocol from 6/12 (week 5)

Notes/Deviations

Retrieved 8 vials of second set of Scott-Simanis competent cells (XL1 Blue from 6/8/18) from -80 and brought upstairs on ice (200 μL in each tube)
DNA added: BBa_J23100, BBa_J23110, BBa_J23114, BBa_K864400, BBa_K1472601, BBa_K1316012, BBa_590031, BBa_590032
Heat shocked four at a time- tried to fit all 8 in at once but four hands wouldn’t fit in the water bath to hold them all so 4 were in briefly then taken out and put back on ice until the the first four were finished and this could have caused problems
Used 2YT media (richer than Ψb)
Did not originally add 800μL media to tube with BBa_590031 tube and did not realize until it was on heat block for ~10 minutes and then added so this could have caused problems
Plated out 200 μL of cells + media except for BBa_K1472601 and BBa_J23100 which we did two plates, one 200 μL and one 300 μL
Trouble keeping flame going (low on butane)
Put in 37 °C incubator at 4:20 p.m.

June 20, 2018

SS Competent Cells Trasformation Test Day 2

Peter

Notes

Observed growth on both the 200 μL and 300 μL plate for BBa_K1472601 and BBa_J23100, the BBa_J23110 plate, and the BBa_J23114 plate
~ 15 colonies (give or take 5) on average per plate
Will redo transformations of parts that were not successful (BBa_K864400, BBa_K1316012, BBa_590031, BBa_590032)

June 20, 2018

SS Competent Cells Trasformation Test Day 1 Repeat

Liz, Toni

Also resuspended BBa_B0015 from 2018, Plate 3, Well 3F (Cam resistance)

Retrieved 5 vials of SS competent cells from -80°C downstairs on ice

competent cells from 6/8/18

Let cells thaw at room temp for ~1 min

Added 1 μL DNA (K1316012, K590032, K864400, K590031, BBa_B0015) to each tube

Competent cell tube for K590031 was empty, had to go downstairs and get another tube so everything was done 6.5 minutes late

Chilled on ice for 30 minutes

K590031 started ice at 6:36 on stopwatch and went till 36:40

Heat shock in 42°C water bath for 45s (tubes held in water bath)

Added 800μL 2xYT media to each tube

Media prewarmed in warm room

Incubated at 37°C on heat block for 60 min

Heat block temporarily dipped to 35°C then went back up to 37°C
Flick tubes every 10 min
K1316012, K590032, K864488, BBa_B0015 in at 2:32, K590031 in at 2:35

Tubes removed from heat block and spun down at 6800g for 3 minutes ~800μL supernatant removed and discarded and cells resuspended in remaining media Cells plated and put in 39°C overnight (in at ~4ish)

Flame ran out of butane

June 21, 2018

SS Competent Cells Trasformation Test Day 1

Peter, Ryan

See protocol from 6/12 (week 5)

Notes/Deviations:

Retrieved 4 vials of second set of Scott-Simanis competent cells (XL1 Blue from 6/8/18) from -80 and brought upstairs on ice (200 μL in each tube)
DNA added: BBa_K864400, BBa_K1316012, BBa_590031, BBa_590032
Used 2YT media (richer than Ψb)
Plated out 200 μL of cells per plate
Used beads in hood instead of flame w/ pasteur pipette

June 22, 2018

Making Media for iGEM Competent Cells

Shannon and Ryan

Made 1L SOB and 1L CCMB80 according to the iGEM protocol.

SOB

Reagent	Amount (g)	Measured Amount (g)
Yeast extract	5	5.0006
Tryptone	20	20.0009
NaCl	0.584	0.5846
KCl	0.186	0.1886
MgSO₄ * 6H₂O	4.93	4.9333

CCMB80

Reagent	Amount (g)	Measured Amount (g)
KOAc	5mL of 1M stock (0.49g)	0.4938
CaCl₂ anhyd.	4.44	4.4473
MnCl₂ * 4H₂O	2.0	2.0062
MgCl₂ * 6H₂O	1.0	1.0081
Glycerol (100%)	5mL	5mL

SOB was pH adjusted to pH 7.54
100mL SOB was aliquoted and 1.6078g agar was added and autoclaved, and then used to make 5 plates. The agar did not dissolve when stirred, but did dissolve in the autoclave. These plates were left in the hood for the weekend.
At first, we tried to make only 500mL of CCMB80, but we used 1M HCl, which decreased the pH much too rapidly, bringing it to around pH 5.8 after 1 drop. So, we doubled the amount and used 0.1M HCl. The final pH was 6.35.

June 22, 2018

Glycerol Stocks for Overnight Cultures

Liz

Made 40% glycerol stock (25mL) in blue falcon tube

10mL 100% glycerol
15mL DI water

Store extra in freezer (-20)

Put 0.75mL 40% glycerol into 10 microcentrifuge tubes (2 tubes per overnight culture, labeled as such)

Put 750 microliters of one culture into two different microcentrifuge tubes

Repeat for each culture, should have 2 microcentrifuge tubes per culture
Pipet up and down in culture to mix cells before transferring

Vortexed tubes to mix

Stored in -80 where DH5alpha cells used to be in a blue box

June 25, 2018

Zyppy miniprep

Liz and Peter

Centrifuge tubes labeled:

*need to look this up*

Added 600 microliters culture to labeled 1.5 mL microcentrifuge tubes

Added 100 microliters 7x lysis buffer (blue) and mixed by inverting 4-6x

Turns opaque to clear blue when lysis is done

*time sensitive!!!* add 350 microliters neutralization buffer (yellow) to each tube

Neutralization buffer should be kept cold
Mixture turns yellow and forms yellow precipitate when neutralized
Invert 2-3x after color change

Centrifuged at 16,000rpm for 3 min then again at 11,000rpm for 1 minute

Pellets got disturbed and had to be repelleted)

Supernatant (~900 microliters) transferred into zymo-spin IIN column (don’t disturb cell pellet)

Put column into collection tube and centrifuge 15 s

Discarded flow-through and put column back into collection tube

Added 200 microliters Endo-wash buffer and centrifuged for 30s

Added 400 microliters zyppy wash buffer and centrifuged 1 min

Put column into clean 1.5mL tube and added 30mL zyppy elution buffer

Let sit 1 minute at room temp

Centrifuged 30s to elute plasmid DNA

June 25, 2018

Primer Rehydration

Liz and Peter

Spun down dry tubes

Added 1xTE buffer to make 100 micromolar solutions

primer	Amount TE added (microliters)
SB prep-3P-1	201
SB prep-2Ea	221
fadD_P1F	201
fadD_P1R	231

Vortexed

Stored in -20 degree freezer in primer box

June 25, 2018

SOB plates

Ryan

SOB plates failed/ dried up because they were left in the hood over the weekend (agar was thin and had a wavy texture, we felt it was better to make new plates).
100mL SOB was aliquoted and 1.6 g agar was added
The solution was heated and mixed, and then autoclaved for an hour (set at liquids 30, P03)
The agar did not completely dissolve when stirred, but did dissolve in the autoclave.
This was used to make 5 plates, which were of proper thickness

June 26, 2018

Making new competent JM109 cells

Liz and Peter

Streaking a SOB plate w/ Rachel’s JM109 cells (SOB plates specific to Top10 cells…)
Put in incubator at 1:45 p.m.
Results (6/27): good growth on plate

June 26, 2018

PCR of fadD

Liz, Peter, and Shannon

Thermocycler Settings

Cycle	Time	Temp. (C)
Initial denaturation	30s	98
35 Cycles	10s	98
	30s	61
	20s for fadD (should have been 40 s because 2kb) 50s for backbone	72
Final extension	10 min	72
Hold	indefinitely	4

Notes

For 61C during the 35 cycles, the time was found using NEBTm calculator
fadD was accidently put at 20s instead of 40s at 72C during the 35 cycles
We aren’t sure if fadD was held at 4C because it didn’t feel cold when opened

Add to PCR tube (for fadD):

Component	Amount (µL)
Nuclease-free water	12.2 (20 - sum of other components)
Phusion buffer (HF)	4
10mM dNTPs	0.4
10mM forward primer	1
10mM reverse primer	1
Template (E. coli) DNA	0.56
Phusion polymerase (add last)	0.2
DMSO	0.6

Notes

Made a 10mM stock solution for dNTPs (1µL of dATP, dTTP, dCTP, dGTP)
Diluted the forward and reverse primer to 10µM 100µM(V) = 10µM(1µL)
2x[0.1µL stock, 0.9µL H2O]
For template DNA, needed 40ng, stock was 40ng
40ng x 1µL/71ng = 0.56µL

Procedure repeated for backbone

4 backbones (PSB1A3, PSB1K3, PSB1T3, PSB1C3), put into 4 labeled PCR tubes
For primers use SB Prep-3P-1 and SBPrep - 2Ea
Annealing temp is 69C
Template DNA: 40ng x 1µL/25ng = 1.6 µL = 1.6 µL backbone DNA

Add to each tube:

Component	Amount (µL)
Nuclease free water	11.2
Backbone DNA	1.6
Phusion buffer (HF)	4
dNTPs	0.4
10mM forward primer	1
10mM reverse primer	1
DMSO	0.6
Phusion polymerase (add last)	0.2

Note: thermocycler did not feel cold when taking out, don’t know if this actually worked

June 27, 2018

PCR Purification

Liz and Shannon

Used protocol from PureLink PCR Purification Kit

Pipet 20µL PCR sample (PSB1T3, PSB1K3, PSB1C3, PSB1A3) into individual 1.5mL tubes
Add 80µL Binding Buffer 2 (B2) to each sample (4x volume) - pipette to mix

B2 is used for purifying PCR fragments

Load each solution into individual spin column collection tubes. Centrifuge at 10,000g for 1 min. Discard flow through and re-insert the column into the collection tube.
Add 650 µL wash buffer (W1) (ethanol had already been added). Centrifuge at 10,000g for 1 min. Discard flow-through and re-insert the column into the collection tube.
Re-centrifuge at max speed (16,100) for 3 min.
Using a clean collection tube, add 50µL Elution Buffer (E1) to column. Let sit for 1 min, then centrifuge at max speed for 2 min.

The purified PCR fragments are in the collection tube.

Products were nanodropped:

PSB1T3: 20.1ng/µL
PSB1K3: 7.8ng/µL
PSB1C3: 21.1ng/µL
PSB1A3: 23.3ng/µL

June 27, 2018

JM109 Overnight Culture

Shannon

SOB was not coming to room temp. Fast enough, so it was put in the warm room for about 10 min.

Used at about 21.1C

Put 5mL SOB into 2 round-bottom tubes and used a non-filtered pipette tip to inoculate the tubes with 2 colonies
Put in downstairs shaker overnight
Note: forgot to do this in the hood!

June 27, 2018

PCR of phz from Genomic DNA (1F-1R, 2F-3R, 4F-4R, 6F-6R)

Peter and Liz

Thermocycler Settings:

Cycle	Time	Temp. (C)
Initial denaturation	30s	98
35 Cycles	10s	98
	30s	64.5
	2F-3R: 54 s 1F-1R, 4F-4R, 6F-6R: 25s	72
Final extension	10 min	72
Hold	Indefinitely	4

Notes:

1F-1R: 0.5 kb
2F-3R: 1.8 kb
4F-4R: 0.9 kb
6F-6R: 1 kb

Add to PCR tubes:

Component	Amount (µL)
Nuclease-free water	11.97 (20 - sum of other components)
Phusion buffer (HF)	4
10mM dNTPs	0.4
10mM forward primer	1
10mM reverse primer	1
Template Genomic DNA (Pseudomonas aeruginosa)	0.83 (40 ng - stock is 48.3 ng/ µL)
Phusion polymerase (add last)	0.2
DMSO	0.6

Note: Started machine 2 on a 50 µL reaction for ~3 seconds and then stopped it and within 10 seconds started again for a 20 µL reaction

June 27, 2018

PCR purification and gel of backbones and phz

Liz

Made a gel for PCR products

25mL 1x TAE buffer and 0.25g agarose in flask and microwaved until dissolved
Let flask cool until it could be picked up
Added 2.5µL SYBR Safe
Poured into mold, put in comb, and let harden under ice bin to protect SYBR Safe from light

Purified PCR products (PureLink PCR Purification Kit)

Pipet 20µL PCR sample (1F/1R, 2F/3R, 4F/4R, 6F/6R) into individual 1.5mL tubes
Add 80µL Binding Buffer 2 (B2) to each sample (4x volume) - pipette to mix

B2 is used for purifying PCR fragments

Load each solution into individual spin column collection tubes
Centrifuge at 11,000g for 1 min and discard flow through
Add 650 µL wash buffer (W1) (ethanol had already been added)
Centrifuge at 11,000g for 1 min and discard flow-through
Centrifuge at max speed (16,100) for 3 min.
Add 50µL Elution Buffer (E1) to column and let sit for 1 min
Centrifuge at max speed (16,100) for 2 min.

The purified PCR fragments are in the collection tube

Nanodrop (blanked with elution buffer)

1F/1R: 5.5 ng/µL
2F/3R: 11.5 ng/µL
4F/4R: 6.4 ng/µL
6F/6R: 6.3 ng/µL
Readings were very inconsistent (can we trust the nanodrop?)

Gel electrophoresis

For each sample mixed 4µL water, 1µL loading dye, and 1µL PCR product
Filled rig with 1x TAE buffer then loaded

1	2	3	4	5	6	7	8	9	10
Ladder (2-log)	pSB1A3	pSB1C3	pSB1K3	pSB1T3	1F/1R	2F/3R	4F/4R	6F/6R	Ladder (2-log)

Filled rig with 1x TAE buffer then loaded

1	2	3	4	5	6	7	8	9	10
Ladder (2-log)	pSB1A3	pSB1C3	pSB1K3	pSB1T3	1F/1R	2F/3R	4F/4R	6F/6R	Ladder (2-log)

Gel did not come out

June 28, 2018

PCR troubleshooting

Liz and Peter

Redid PCR protocol for fadD and phz 2F/3R but used new dNTPs

Time		Temp. (C)
fadD	phz 2F/3R	fadD	phz 2F/3R
30s	30s	98	98
10s	10s	98	98
40s	54s	72	72
10 min	10 min	72	72
infinite	infinite	4	4

Annealing temperatures from NEB annealing temperature calculator

Set to 20µL reaction

Labeled PCR tubes fadD and phz 2F/3R

Component	Amount (µL)
	fadD	phz 2F/3R
Nuclease free water	12.2	11.97
Template DNA (40ng)	0.56*	0.83*
Phusion buffer (HF)	4	4
dNTP stock	0.4	0.4
10 mM forward primer	1	1
10mM reverse primer	1	1
DMSO	0.6	0.6
Phusion polymerase (add last)	0.2	0.2

Calculations

fadD: 40ng x 1µL/71ng = 0.56µL
phz 2F/3R: 40ng x 1µL/48.3ng = 0.83µL

New dNTP stock diluted (0.2µL each dNTP and 7.2µL dH₂O) because old one had been thawed/refrozen too many times

Made a gel for PCR products

12.5mL 1x TAE buffer and ~0.125g agarose in flask and microwaved until dissolved

Can’t tell if gel is 1% because scale measures to nearest 0.05g

Let flask cool until it could be picked up
Added 1.75µL SYBR Safe
Poured into mold, put in comb, and let harden under ice bin to protect SYBR Safe from light

June 28, 2018

PCR troubleshooting purification/gel

Liz and Peter

Purified PCR products (PureLink PCR Purification Kit)

Pipet 20µL PCR sample (fadD and phz 2F/3R) into individual 1.5mL tubes
Add 80µL Binding Buffer 2 (B2) to each sample (4x volume) - pipette to mix

B2 is used for purifying PCR fragments

Load each solution into individual spin column collection tubes
Centrifuge at 11,000g for 1 min and discard flow through
Add 650 µL wash buffer (W1) (ethanol had already been added)
Centrifuge at 11,000g for 1 min and discard flow-through
Centrifuge at max speed (16,100) for 3 min
Add 50µL Elution Buffer (E1) to column and let sit for 1 min
Centrifuge at max speed (16,100) for 2 min

The purified PCR fragments are in the collection tube

Nanodrop (blanked with elution buffer)

fadD: 11.2 ng/µL
phz 2F/3R: 11.3 ng/µL

Gel electrophoresis

For each sample mixed 4µL water, 1µL loading dye, and 1µL PCR product
Topped off rig with 1x TAE buffer then loaded
Ran at 50V for 50 min

1	2	3	4	5
Ladder (2-log)	empty	phz 2F/3R	empty	fadD

June 29, 2018

Backbone and phz PCR

Liz and Peter

Parts to PCR (1 PCR tube per sample):

phz 1F/1R (510bp)
phz 4F/4R (946bp)
phz 6F/6R (997bp)
pSB1A3 (2155bp)
pSB1C3 (2070bp)
pSB1K3 (2204bp)
pSB1T3 (2461bp)

Set thermocycler:

Time		Temp. (C)
phz	backbones	phz	backbones
30s	30s	98	98
10s	10s	98	98
30s	30s	64.5	69
25s	50s	72	72
10 min	10 min	72	72
infinite	infinite	4	4

Added to each tube:

Component	Amount (µL)
	phz	backbones
Nuclease free water	11.97	11.2
Template DNA (40ng)	0.83*	1.6*
Phusion buffer (HF)	4	4
dNTP stock	0.4	0.4
10 mM forward primer	1	1
10mM reverse primer	1	1
DMSO	0.6	0.6
Phusion polymerase (add last)	0.2	0.2

Calculations

backbones: 40ng x 1µL/25ng = 0.56µL
phz: 40ng x 1µL/48.3ng = 0.83µL

Tubes kept on ice and vortexed before putting into thermocycler

Gel electrophoresis

For each sample mixed 4µL water, 1µL loading dye, and 1µL PCR product
Filled rig with 1x TAE buffer then loaded

1	2	3	4	5	6	7	8	9	10
Ladder (2-log)	phz 1F/1R	phz 4F/4R	phz 6F/6R	Ladder (2-log)	pSB1A3	pSB1C3	pSB1K3	pSB1T3	Ladder (2-log)

Gel didn’t get imaged correctly first time so redid gel on 7/2/2018:

1	2	3	4	5	6	7	8	9
Ladder (2-log)	pSB1A3	pSB1C3	pSB1K3	pSB1T3	phz 4F/4R	phz 6F/6R	phz 1F/1R	Ladder (2-log)

Ran at 50V for 30+20 min (time in between because didn’t know current stops when timer does)

July

July 2, 2018

iGEM Competent Cell Prep, Day 1

Liz

Going to try with 1 tube JM109 cells and 1 tube NEB Gibson cells

Need to use all detergent-free glassware and rinsed plasticware

Autoclaved glassware filled ¾ of way with dH₂O

Put 250mL SOB media into each of two 500mL flasks (labeled JM109 and NEB cells)

Measured/poured in hood because no antibiotics in SOB
Put media into warm room about 20 minutes before pouring, not sure if this is necessary

Put 1 tube thawed JM109 cells into JM109 flask and 1 tube thawed NEB Gibson cells into NEB flask and covered with foil

Thawed tubes in hand
Pipetted cells into media (not competent yet so shouldn’t kill cells)

Put ice in bottom of ice bucket and left flasks of cells in ice bucket overnight

Want OD₆₀₀ of 0.3 (should be about 16 hrs at 20 degrees C)
Start at 5:00pm

July 3, 2018

iGEM Competent Cell Prep, Day 1

Liz, Shannon

Pre-chilled 2 flat-bottomed centrifuge bottles and preset centrifuge for 4 degrees C

Checked OD₆₀₀ of overnights (want at 0.3):

Time	7:38	8:03	8:39	9:15	9:56	10:30	11:18	11:52	12:34
JM109	0.005	0.006	0.017	0.015	0.026	0.054	0.113	0.179	0.294
NEB cells	0.015	0.016	0.022	0.020	0.024	0.028	0.039	0.055	0.081

After first measurement, kept in warm room
Gave up on NEB cells (not growing)

Put equal amounts JM109 culture into two flat-bottomed centrifuge bottles
Centrifuge for 3000g at 4 degrees C for 10 min
Decant supernatent
Resuspend in 80mL ice cold CCMB80 buffer

40mL each tube
Resuspend in 20mL then pipet other 20mL against walls

Incubate on ice 20 min
Centrifuge at 3000g at 4 degrees C for 10 min
Decant supernatent
Resuspend pellet in 10mL ice cold CCMB80 (10mL is total, put 5mL in each bottle then combined bottles)
Was supposed to check OD of 800μL SOC and 200μL culture using spectrophotometer (blank with 800μL SOC and 200μLCCMB80) then adjust to 1.0-1.5 using CCMB80 but OD started at 0.532 so just skipped this step
Incubated on ice (was supposed to be 20 minutes but ended up being much longer because was trying to figure out the protocol)
Aliquotted 50μL per tube over liquid nitrogen (suspended over top to be cold but not too cold like flash freezing)

Made two batches because liquid nitrogen evaporated

Stored in -80 freezer

July 3, 2018

fadK PCR

Liz

Resuspended and diluted primers

1F: added 471μL to resuspend, to dilute to 10mM added 1μL resuspended primer and 9μL milliQ water
1R: added 339μL to resuspend, to dilute to 10mM added 1μL resuspended primer and 9μL milliQ water

Thermocycler Settings

Cycle	Time	Temperature (degrees Celsius)
Initial denaturation	30s	98
35 cycles	10s	98
	30s	63
	40s	72
Final extension	10 min	72
Hold	Infinite	4

Added to PCR tube:

Component	Amount (µL)
Nuclease-free water	12.2 (20 - sum of other components)
Phusion buffer (HF)	4
DMSO	0.6
10mM forward primer	1
10mM reverse primer	1
dNTPs	0.4
Template (E. coli) DNA	0.56
Phusion polymerase (add last)	0.2

July 5, 2018

fadK PCR purification and Nanodrops

Liz, Sricharan

Used protocol from 6/28 (1 PCR tube for fadK)

Ran 10 Nanodrops (blanked with elution buffer (Buffer E1) from Invitrogen Purif Kit) of PCR purif (4 phz parts, fadD, fadK, 4 linear plasmid backbones)
All ~10-15 ng/µL, good characteristic curves

Sample Name	Conc. (ng/µL)	260/280	260/230
phz1F-1R Purif	13.31	2.04	1.75
phz2F-3R Purif	11.83	1.89	1.53
phz4F-4R Purif	15.53	1.97	1.77
phz 6F-6R Purif	10.65	2.13	1.46
fadD Purif	8.03	1.79	2.49
fadK Purif	14.49	1.69	1.84
pSB1C3 Purif	15.01	2.01	1.72
pSB1T3 Purif	15.56	1.91	1.70
pSB1K3 Purif	12.88	1.94	1.99
pSB1A3 Purif	10.55	2.11	1.78

July 5, 2018

resuspension of phenazine gBlocks

Liz

Needed to resuspend phz4-5 and start of phz for gibson assembly

Centrifuged for 5s to make sure all of product is in bottom of tube

Added 1x TE buffer to get concentration to 10ng/μL

Phz4-5 (1000ng)
1000ng x 1μL/10ng = 100μL
Start of phz (500ng)
500ng x 1μL/10ng = 50μL

Vortexed

Incubated in hat block at 50 degrees C for 20 min

Vortexed and centrifuged

July 5, 2018

phz gibson PCR and fadD/fadK secondary PCR

Liz, Sricharan

Thermocycler Settings

	Phz gibson		fad
	Time	Temperature (℃)	Time	Temperature (℃)
Initial denaturation	30s	98	30s	98
35 cycles	10s	98	10s	98
	30s	61	30s	72
	60s	72	40s	72
Final extension	10 min	72	10 min	72
Hold	infinite	4	infinite	4

Add to PCR tubes:

Component	Amount (µL)
	Phz gibson	fadD	fadK
Nuclease-free water	11.3 (20 - sum of other components)	7.8 (20 - sum of other components)	10.04 (20 - sum of other components)
Phusion buffer (HF)	4	4	4
DMSO	0.6	0.6	0.6
10mM forward primer	1 (1F)	1 (fadD bad start)	1 (fadD bad start)
10mM reverse primer	1 (1R)	1 (fadD bad end)	1 (fadD bad end)
dNTPs	0.4	0.4	0.4
Template (E. coli) DNA	1.5	5.0	2.76
Phusion polymerase (add last)	0.2	0.2	0.2

Gibson Assembly using the NEB kit … wanted 0.2pmol of DNA total (kit specifies 0.2-1pmol for 4-6 pieces)

Start of phz (gBlock)

0.05pmol x 50µL/1.950pmol = 1.3µL

phz 1F-1R Purif

0.05pmol x 157,303pg/1pmol x 1ng/1000pg x 1µL/13.3ng = 0.59µL

phz 4-5 (gBlock)

0.05pmol x 100µL/2.057pmol = 2.4µL

phz 2F-3R Purif

0.05pmol x 561,368pg/1pmol x 1ng/1000pg x 1µL/11.8ng = 2.4µL

For Gibson mix, mixing total volume of 6.69 µL fragments + 10 µL NEB Gibson Master Mix + 3.31 µL dH₂0

Above solution incubated at 50°C for 60 min then at 4°C for infinite hold.

July 9, 2018

Resuspending G-Blocks

Shannon

Resuspended phz 1-2, cymA, mtr start, and tesA using TB
Centrifuge briefly to make sure all products were at the bottom of the tube

Note: Forgot to do this for phz 1-2 (ex. Phz 1-2 had 500ng in original tube, so 500ng x 1µL/10ng = 50µL)

G-Block	Amount of TB (µL)
Phz 1-2	50
cymA	100
Mtr start	25
tesA	100

Vortex briefly
Incubate at 50C for 20 min in heat block

Note: forgot to pre-heat heat block so they sat at room temp for a few minutes

Briefly vortex and centrifuge
Stored at -20C

July 9, 2018

Restriction Digest

Shannon

Used benchling protocol to digest fadD, tesA, cymA, psb1C3, and mtr start
All enzymes checked on nebcloner.neb.com/#!/redigest for incubation temperatures and “time-saver” (they can be incubated at 37C for 10 minutes rather than 1hr) and to verify that CutSmart could be used as the buffer
Add reagents in the order: water → buffer → DNA → enzymes
fadD/tesA/cymA digested with ecoRI and pstI

Component	Amount (µL)
ecoRI	1
pstI	1
CutSmart	2
DNA	5.8 µL for fadD
	10 µL for tesA and cymA
dH₂O	10.2 µL for fadD 6µL for tesA and cymA

Calculations:

For water: 20 µL - volume of all other components
DNA: wanted 100ng, so calculated volume ex. 100ng tesA x 1µL/10ng = 5.8µL

Psb1C3 digested with dpnI, ecoRI, and pstI

Component	Amount (µL)
dpnI	1
ecoRI	1
pstI	1
CutSmart	2
DNA	4
dH₂O	13

Mtr start digested with ecoRI, bspHI

Component	Amount (µL)
ecoRI	1
bspHI	1
CutSmart	2
DNA	10
dH₂O	6

Centrifuge briefly
Heat block at 37C for 10min
Heat block at 80C for 15min (heat-kill enzymes)
Store at 20C

July 9, 2018

fadK, phz gibson PCR (try 2), phz Beg PCR Assembly

Liz, Sricharan

No bands on gel for fadK and phz gibson 1 (7/15) so redid PCR

Notes/changes:

Did two tubes for phz (one uses HF buffer and other uses GC buffer)
Phz 35 cycles time 3 changed to 45s because doing PCR assembly at the same time

Also tried to use PCR assembly instead of Gibson for phz Beg

Just add phz 1F-1R, phz 4-5, and phz 2F-3R to PCR mix and they should assemble together in theory, Phusion does all the gaps
Need 40ng total template (later realized this should be less, 40ng is for genomic DNA)
Since construct is 300bp, this is ~0.02 pmol - using quantities from 7/5/2018

Followed PCR purification protocol from 6/28

Nanodropped pure products

pure_fadD: 17.24 ng/µL
pure_fadK: 3.57 ng/µL
pure_fadK_try2: 4.81 ng/µL
pure_phzgibson1: 17.04 ng/µL

Lane	1	2	3	4	5
Sample	Ladder	fadD PCR Purif	fadK PCR Purif	Phz Beg Gibson PCR

On a 1% agarose gel in TAE with SYBRSafe, as usual, run at 50V for 50min

July 10, 2018

Ligation of cymA, tesA, and fadD

Shannon and Toni

Did a ligation to insert cymA, tesA, and fadD, separately, into psB1C3 backbones
Using ligation protocol on benchling, calculated ng of insert dna needed via link

Sample	Insert DNA Length	Vector DNA Length	Vector DNA Mass	Insert DNA Mass	Ratio
tesA	800kb	2070kb	30ng	34.78ng	3:1
Fad D	1713kb	2070kb	30ng	49.65ng	2:1
cymA	773kb	2070kb	30ng	33.61ng	3:1

tesA	fadD	cymA	Added
4 µL	1.1 µL	4.3 µL	dH₂O
2 µL	2 µL	2 µL	T4 buffer
6 µL	6 µL	6 µL	psB1C3 backbone
7 µL	9.9 µL	6.7 µL	DNA insert
1 µL	1 µL	1 µL	T4 ligase

Total volume of soln: 20ul; 100ng/20ul (20ng/ul) of stock vector/inserts
All reagents kept on ice and added in the order listed
Incubated at RT for 10 min
Incubated at 65C for 20 min
Placed immediately into -20C

July 10, 2018

PCR purification

Liz

Samples (4):

Phz gibson 1 product

With HF buffer
With GC buffer

fadK (secondary PCR)
Phz assembly (PCR assembly)

Followed protocol from 6/28

Nanodropped purified product

Phz gibson HF: 5.93 ng/µL
Phz gibson GC: 10.77 ng/µL
fadK: 9.28 ng/µL
Phz assembly: 20.11 ng/µL

Ran gel at 50V for 42 min

1	2	3	4	5
ladder	Phz gibson GC	Phz gibson HF	fadK	Phz PCA

July 10, 2018

MIT Competent Cells

Liz, Sricharan, Shannon, Toni

Using protocol given to us at NEGEM by Brian Teague of MIT

2 5mL OCs of JM109 were put in 2xYT the day before

Put 800µL JM109 overnight cultures into 400mL LB broth and put into shaker (37 degrees C, 250rpm) at 9:30am

Making transformation buffers (TfbI, 150mL; TfbII, 20mL):

	TfbI Needed	TfbI Actual	TfbII Needed	TfbII Actual
RbCl	1.8g	1.802g	0.024g	0.0235g
MnCl₂	1.5g	1.508g	-	-
CaCl₂	0.166g	0.171g	0.166g	0.166g
KOAc	0.442g	0.448g
MOPS	-	-	0.042g	-
Glycerol	22.4mL	Measured w/grad cyl	3.0mL	Measured with serological pipet

Checked OD:

Time	11:51	13:15
OD₆₀₀	0.156	>1

Failure - try again tomorrow; using TfbI and TfbII made today, which is not ideal but we’re also running out of RbCl

July 11, 2018

MIT Competent Cells

Liz, Sricharan, Peter

2 5mL OCs of JM109 were put in LB the day before
Put 800µL JM109 overnight cultures into 380mL LB broth and put into shaker (37 degrees C, 250rpm) at 7:30am

Want OD₆₀₀ of 0.5-0.7

Checked OD:

Time	9:25	10:02	10:22	10:41	10:46	10:54
OD₆₀₀	0.056	0.164	0.289	0.416	0.506	0.565

July 11, 2018

Transformation of Competent Cells

Peter and Shannon

Did a transformation (iGEM protocol) with GFP to test that the cells are competent
Pre-chill 3 1.5mL tubes of 50µL competent cells - kept on ice while labeling/pipetting
Add 10, 50, and 100 pg/µL of RFP construct to tubes
Incubate on ice for 30 min
Heat shock for 45 sec in 37C water bath
Incubate 5 min on ice
Add 950 pre-warmed 2YT to each tube
Incubate 1 hr at 37C on heat block
Note: forgot to do a control - will do tomorrow

July 12, 2018

PCR of FadK using GC buffer

Shannon

Thermocycler Settings

Phase	Time (sec)	Temp. (C)
Initial denaturation	30	98
35 cycles	10	98
	30	72
	40	72
Final extension	10	72
Hold	∞	4

Add to tubes:

Component	Amount (µL)
dH₂O	10
GC buffer	4
DMSO	0.6
10mM forward primer (bad)	1
10mM reverse primer (bad)	1
dNTP mix	0.4
fadK purified PCR (template)	2.76
phusion	0.2

All components were kept on ice

July 12, 2018

iGEM Transformations

Liz, Sricharan

Tubes:

cmmA-H (biobrick, resuspended in 10µL milliQ water and stored in Registry Parts 2018 box in -20C)
RFP 25s HS
RFP 45s HS
RFP 60s HS
RFP 90s HS

Thawed competent cells on ice (~6 min)
Put 1µL DNA into each corresponding tube
Incubated on ice for 30 min

Heat shocked in 42C water

RFP 25s HS: 25s
RFP 45s HS, ccmA-H: 45s
RFP 60s HS: 60s
RFP 90s HS: 90s

Incubated on ice for 5 min
Added 450µL SOC media to each tube
Incubated in shaker (37C, 250rpm) for 1 hr
Spun down cells at 6800g for 3 min and discarded ~350-400mL
Resuspended cells and plated remaining 100µL in hood using pipet tip to spread
Incubated overnight at 37C (put in ~4:15)
Took plates out ~9:00 7/13 - it worked (only for ccmA-H)!!

July 12, 2018

Gibson of phz 1-2, 4F-4R, 6F-6R

Peter, Ryan

Set up the following reaction on ice:

phz 1-2 (gBlock): 0.045pmol x 50µL/1.180pmol = 1.91µL
phz 4F-4R Purif: 0.045pmol x 614,900pg/1pmol x 1ng/1000pg x 1µL/15.53ng = 1.78µL
phz 6F-6R Purif: 0.045pmol x 584,050pg/1pmol x 1ng/1000pg x 1µL/10.65ng = 2.74µL

For Gibson mix, mixing total volume of 6.43 µL fragments + 10 µL NEB Gibson Master Mix + 3.57 µL dH₂0

Above solution incubated at 50°C for 60 min then at 4°C for infinite hold.

July 12, 2018

PCR Cleanup of FadK

Shannon

Combine 80µL B2 buffer (4x the amount of PCR sample) to 20µL PCR sample
Transfer to collection tube, centrifuge at 10,000 rpm for 1 min. Discard flow-through. Re-insert column into collection tube.
Add 650µL wash buffer with ethanol. Centrifuge at 10,000 rpm for 1 min. Discard flow-through. Re-insert column into collection tube.
Centrifuge at max speed (16,100rpm) for 3 min. Discard flow-through. Insert column into a clean 1.7mL elution tube.
Add 50µL elution buffer to center of column.
Incubate at room temperature for 1 min.
Centrifuge at max speed for 2 min.
The elution tube now contains purified PCR product!
Store at -20C.

July 13, 2018

PCR of Gibson assembly from 7/12 (Phz 4F-4R, Phz 1-2, Phz 6F-6R)

Peter, Ryan

Thermocycler Settings

Phase	Time (sec)	Temp. (C)
Initial denaturation	30	98
35 Cycles	10	98
	30	67
	78	72
Final Extension	10	72
Hold	∞	4

Component	Amount (µL)
dH₂O	10.15
GC buffer	4
DMSO	0.6
10mM forward primer (bad)	1
10mM reverse primer (bad)	1
dNTP mix	0.4
DNA Template from Gibson Assembly of	2.65
phusion	0.2

All components were kept on ice

July 13, 2018

Gel Electrophoresis of Purified FadK PCR

Shannon

Prepare gel

Combine 15 mL 1x TAE and 0.125g agarose (weigh between 0.10 and 0.15 because our scale isn’t that accurate) in a small erlenmeyer flask
Microwave until dissolved
Cover with foil, let cool until flask isn’t too hot to handle
Add 1.25µL Sybr Safe, swirl
Pour into a small mold, add comb
Let harden

July 13, 2018

LB Broth

Liz

500mL Broth

Component	Amount (g)	Measured (g)
tryptone	5	5.05
Yeast extract	2.5	2.50
NaCl	5	5.00

QSed to just under 500mL
Put in stir bar and stirred/heated for 15 min
QSed to 500mL in graduated cylinder
Autoclaved on liquids 30 then stored in 4C

July 13, 2018

PCR Cleanup of Phz(4F-6R Gibson)

Ryan and Peter

Combine 80µL B2 buffer (4x the amount of PCR sample) to 20µL PCR sample
Transfer to collection tube, centrifuge at 10,000 rpm for 1 min. Discard flow-through. Re-insert column into collection tube.
Add 650µL wash buffer with ethanol. Centrifuge at 10,000 rpm for 1 min. Discard flow-through. Re-insert column into collection tube.
Centrifuge at max speed (16,100rpm) for 3 min. Discard flow-through. Insert column into a clean 1.7mL elution tube.
Add 50µL elution buffer to center of column.
Incubate at room temperature for 1 min.
Centrifuge at max speed for 2 min.
The elution tube now contains purified PCR product!
Store at -20C.
Nanodroped @ 16.7 ng/µL

July 13, 2018

Gel of PCR’s for Phz 4F-6R, fadK Primary and Secondary PCR’s

Peter, Ryan

Wells

1	2	3	4	5
2 Log Ladder	PHz 4F-6R (Gibson) purif.	fadK Primary purif. (7/5/18)	fadK secondary purif. (7/9/18)	fadK secondary purif. (7/12/18)
-6 µL ladder	-2 µL purif. PCR -3 µL MilliQ DH2O -1 µL Loading Dye	-5 µL purif. PCR -1 µL Loading Dye	-5 µL purif. PCR -1 µL Loading Dye	-5 µL purif. PCR -1 µL Loading Dye

Gibson worked… two visible bands, one a little less than 3 kb according to the ladder (full gibson should be 2.588 Kb) and one band that is probably both 4F-4R and 6F-6R that didn’t assemble are about both ~ 1 Kb
Secondary fadK most likely didn’t work because PCR used ~ 40 ng from Primary PCR so that is probably what is visible in this gel (also same length as primary PCR)

July 16, 2018

Secondary PCR of fadD and fadK

Ryan, Shannon

Set thermocycler:

Phase	Time (sec)	Temp. (C)
Initial denaturation	30	98
35 cycles	10	98
	30	67
	40	72
Final extension	10 minutes	72
Hold	∞	4

Add to tubes:

Component	fadD Amount (µL)	fadK Amount (µL)
dH₂O	12.6	12.5
Phusion buffer (HF)	4	4
DMSO	0.6	.6
10mM forward primer (bad)	1	1
10mM reverse primer (bad)	1	1
dNTP mix	0.4	.4
Template DNA	.2	.3
phusion	0.2	.2

All components were kept on ice

July 16, 2018

Restriction Digest of Secondary fadD and fadK PCRs

Liz

fadD and fadK:

Component	Amount
EcoRI (HF)	1µL
PstI (HF)	1µL
CutSmart	2µL
DNA template	100ng
	fadD: 100ng x 1µL/14.3ng = 6.99µL
	fadK: 100ng x 1µL/19.9ng = 5.02µL
Water	To 20µL
	fadD: 9.00µL
	fadK: 10.97µL

Order: water, buffer, DNA, enzymes
Spin down in tabletop centrifuge
Heat blocked at 37C for 25 min
Heat blocked at 65C for 15 min

Backbone:

Component	Amount
BpsHI	1µL
EcoRI (HF)	1µL
PstI (HF)	1µL
CutSmart	2µL
DNA template	100ng
	fadD: 100ng x 1µL/14.3ng = 6.99µL
	fadK: 100ng x 1µL/19.9ng = 5.02µL
Water	To 20µL
	fadD: 9.00µL
	fadK: 10.97µL

Order: water, buffer, DNA, enzymes
Spin down in tabletop centrifuge
Heat blocked at 37C for 25 min
Heat blocked at 80C for 15 min
This digest was wrong because DnpI should have been used for the backbone instead of BpsHI

July 16, 2018

Gel Electrophoresis of Purified FadK, FadD PCR

Ryan

Prepare gel

Combine 15 mL 1x TAE and 0.125g agarose (weigh between 0.10 and 0.15 because our scale isn’t that accurate) in a small erlenmeyer flask
Microwave until dissolved
Cover with foil, let cool until flask isn’t too hot to handle
Add 1.25µL Sybr Safe, swirl
Pour into a small mold, add comb
Let harden

Wells:

1	2	3	4	5
2 Log Ladder	fadD PCR2	fadD PCR0	fadK PCR2	fadK PCR0
-6 µL ladder	-1 µL purif. PCR -4 µL MilliQ DH2O -1 µL Loading Dye	-1 µL purif. PCR -4 µL MilliQ DH2O -1 µL Loading Dye	-1 µL purif. PCR -4 µL MilliQ DH2O -1 µL Loading Dye	-1 µL purif. PCR -4 µL MilliQ DH2O -1 µL Loading Dye

July 16, 2018

Transformation with RbCl JM109 Cells

Sricharan, Peter

Transforming:

BBa_J04450 (RFP), BBa_B0015 (DoubleTerminator), BBa_K590025 (PetroBrick), BBa_K1433005 (Lambda Red), BBa_I718008 (Inducible Cre-generator), BBa_K1316012 (Mtr + T7 Lac O.), TesA (from ligation), CymA (from ligation), BBa_K917006 (ccmA-H)

Grab 50μL aliquots of RbCl Uber-Competent Cells from -80° C freezer, let thaw on ice for ~3-4 minutes.
Add 1μL of plasmid from iGEM kit (or 2 μL of plasmid from ligation product) to the tube with cells.
Incubate cells on ice for 30 minutes.
Heat shock tubes at 42° C for 45s.
Incubate on ice again for 5 minutes.
Add 450μL SOC media to each tube (9 x volume) and incubate on the shaker at 37° C and 250rpm for 1 hour.
Spin down cells at ~6800rpm for 3 minutes, remove 370μL of supernatant, resuspend cell pellet in the remaining media, and pipet that onto the plate
Use P200 to spread cells across plate
Leave plates upside-down (with agar on top) in incubator (at 37° C) overnight for 16-18 hours.

Notes:

Added 2 μL pf DNA for TesA & CymA because from ligation reaction so not as much DNA as a prepared BioBrick
Heat-shocked ccmA-H (BBa_K917006) in 5 different tubes for 30, 45, 60, 75, and 90 seconds (one time per tube)
Added 900 μL of soc media for tube 4 because was 100 μL aliquot of cells
Used Amp plate for BBa_I718008 because plasmid has Amp resistance

Results (7/17/18):

Part/ Vector	Transformation Result
BBa_J04450 (RFP)	-No growth on plate
BBa_B0015 (Double Terminator)	-No observable cell pellet when spun down -No growth on plate
BBa_K590025 (PetroBrick)	-Good growth (do O.C. on 7/17)
BBa_K1433005 (Lambda Red)	-Good growth (do O.C. on 7/17)
BBa_I718008 (Inducible Cre-generator)	-Good growth (do O.C. on 7/17)
BBa_K1316012 (Mtr + T7 Lac O.)	-No observable cell pellet when spun down -No growth on plate
TesA (from ligation)	-No growth on plate (Ligation enzymes can be a problem)
CymA (from ligation)	-No growth on plate (Ligation enzymes can be a problem)
BBa_K917006 (ccmA-H)	-30 sec: works best -45 sec: works well -60 sec: works okay -75 sec: no visible pellet but grew a few colonies -90 sec: no visible pellet or growth on plate (probably heat-killed)

July 17, 2018

Ligation of fadD and fadK

Liz

*didn’t work because digested backbone wrong
fadD:
Insert DNA: 1689 bp
Vector length: 2070 bp
Vector mass: 25 ng
NEB Ligation Calculator: 2:1 → 40.80ng x 20L

July 17, 2018

Gel Electrophoresis of Purified FadK, FadD PCR

Ryan

Prepare gel

Combine 15 mL 1x TAE and 0.125g agarose (weigh between 0.10 and 0.15 because our scale isn’t that accurate) in a small erlenmeyer flask
Microwave until dissolved
Cover with foil, let cool until flask isn’t too hot to handle
Add 1.25µL Sybr Safe, swirl
Pour into a small mold, add comb
Let harden

Wells:

1	2	3	4	5
2 Log Ladder	fadD PCR2	fadD PCR0	fadK PCR2	fadK PCR0
-6 µL ladder	-1 µL purif. PCR -4 µL MilliQ DH₂O -1 µL Loading Dye	-1 µL purif. PCR -4 µL MilliQ DH₂O -1 µL Loading Dye	-1 µL purif. PCR -4 µL MilliQ DH₂O -1 µL Loading Dye	-1 µL purif. PCR -4 µL MilliQ DH₂O -1 µL Loading Dye

No bands visible on any part of the gel:

July 17, 2018

Gel Extraction of phz 4F-6R

Sricharan

Onto a 1% agarose gel with SYBR Safe, loaded (with 6X Purple Loading Dye from NEB)

1	2	3	4	5
2-log ladder	phz 1F-3R PCR (GC Buffer)	‘tesA(L109P) ligation	phz 4F-6R PCR	Same as Lane 4
6µL ladder	5µL item 1µL dye	1µL item 4µL dH₂O 1µL dye	15µL item 3µL dH₂O

Note, wells 2 and 3 had blue stain from tape (we tried to tape together these two wells to do gel extraction but it didn’t work very well).

Cut bands at 2.6kb on last two lanes out using tape (marked with arrows delineating top of gel, bottom of gel, and location of band to be excised) and wax paper + razor
Weighed cut bands: 0.0840g, used Zymoclean Gel DNA Recovery Kit to clean up DNA. Only deviation from protocol was centrifuged ~16K rpm to elute 30µL buffer from Invitrogen PCR Pure Kit.

Nanodrop Results:

Conc: 2.8 ng/µL
260/280: 1.23
260/230: 0.05

July 17, 2018

LB + Chloramphenicol Plates Preparation

Ryan

Preparing Chloramphenicol LB plates (24)

Collected and measured out ingredients for LB media and added to bottle
Q/s’d with dH₂0 to 400 and shook bottle to dissolve ingredients
Stirred in beaker for 30 min
Poured into grad cylinder to finish Q/s to 500 mL
Poured back into bottle and stirred for ~ a minute
Autoclaved on S03 setting for 30 minutes (autoclaving less than a liter of liquid)
Added 0.5 mL of chloramphenicol (1000x, 35 mg/mL) to solution
Put them in the hood with the plates half open w/ agar side on the bottom to get rid of condensation

**OOPS put in 35 microliters instead of .5 mL

July 17, 2018

Transformation with RbCl JM109 Cells

Liz, Peter

**See Protocol from 7/16/18**

Transforming failed transformations from 7/16:

BBa_J04450 (RFP), BBa_B0015 (DoubleTerminator), BBa_K1316012 (Mtr + T7 Lac O.), TesA (from ligation + now purified w/ PCR purif), CymA (from ligation + now purified w/ PCR purif), BBa_K917006 (ccmA-H)

***See protocol from 7/16***

Notes/ Deviations:

CymA and TesA have been purified w/ PCR purif.
Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube (only one tube for the blank)
Heat-shocked for 30 seconds based on results from transformations of ccmA-H
After resuspending, pooled secondary tube w/ primary tube if any cells (next time pool before spinning)
All except blank had one tube w/ obvious pellet after first spin
BBa_J04450 (RFP), BBa_B0015 (Double terminator), and CymA secondary tubes w/ no original pellet had slightly visible pellet after second spin. Were pooled with primary tube after resuspension
Not pellet for 6 so didn’t bother plating

Results (7/18/18):

Part/ Vector	Transformation Result
BBa_J04450 (RFP)	-No growth on plate
BBa_B0015 (Double Terminator)	-One colony (Do O.C. on 7/18)
BBa_K1316012 (Mtr + T7 Lac O.)	-No growth on plate
TesA (from ligation + now purified w/ PCR purif)	-No growth on plate
CymA (from ligation + now purified w/ PCR purif)	-One colony (Do O.C. on 7/18)

July 17, 2018

Overnight Cultures of successful transformations from 7/16/18

Peter, Shannon

Overnight Cultures:

BBa_K590025 (PetroBrick)
BBa_K1433005 (Lambda Red)
BBa_I718008 (Inducible Cre-generator)
BBa_K917006 (ccmA-H)

Results (7/18/18):

They worked: proceed to mini-prep and glycerol stocking

July 18, 2018

Nanodrops and a Gel: Miniprepped Plasmids, Gel Extraction PCR of 4F-6R, Gel Extraction of 1F-3R

Sricharan

Some OCs were grown - Shannon miniprepped and Liz made glycerol stocks; I Nanodropped

Item	Concentration (ng/µL)	260/280	260/230
I718008 MP	85.7	1.75	1.28
K590025 MP	51.7	1.73	0.85
K1433005 MP	53.9	1.74	1.37
K917006 MP	175.1	1.88	2.15
Phz 4F-6R GelExtr PCR	30.9	1.39	-0.82

Ran a gel:

Lane	1	2	3	4	5	6
Item	2-log ladder	Poked hole	K143305 MP	I718008 MP	K917006 MP	PCR of Gel Extr of phz 4F-6R
Amt (µL)	6	N/A	5+1 dye	5+1 dye	5+1 dye	2+3 dH₂O+1 dye

7	8	9	10	11	12
2-log ladder	phz 1F-3R GC PCR	phz 1F-3R GC PCR	empty	K590025 MP	2-log ladder
6	15 + 3 dye	15 + 3 dye		5 + 1 dye	6

Note, items in lanes 3, 4, 5, 11 digested with PstI.
Dye is 6X Purple Loading Dye from NEB.
Liz gel extracted bands 8 and 9 at desired size (~3kb)

PCR of these bands:
To PCR tube on ice:

dH₂O	11.8 µL
Phusion Buffer (GC)	4 µL
DMSO	0.6 µL
10µM forward primer (1F)	1 µL
10µM reverse primer (3R)	1 µL
10µM dNTPs	0.4 µL
Template DNA	1 µL
Phusion polymerase	0.2 µL

Cycle is based on Phusion polymerase protocol:

	Time (s)	Temp (°C)
Initial Denaturation	30	98
35 Cycles	10	98
	30	61
	60	72
Final Extension	600	72
Hold	Infinite Hold	4

July 18, 2018

Miniprep of overnight cultures from 7/17/2018

Shannon

Used Zyppy Miniprep Kit and instructions from the manufacturer’s website
Overnight cultures of: K590025, K1433005, I718008, and K917006
Add 600µL of each bacterial culture to separate 1.5mL tubes
Add 100µL 7x lysis buffer, mix by inverting about 6 times

Solution should turn from opaque blue to clear blue

Add 350µL cold neutralization buffer (yellow), mix by inverting

Solution should turn yellow

Centrifuge at 13,000g for 3 min
Transfer supernatant (about 900µL) into column, place column in collection tube
Centrifuge for 15 seconds
Discard flow-through, put column back into the same collection tube
Add 200µL endo wash buffer, centrifuge 20 seconds
Add 400µL Zippy wash buffer to column. Centrifuge 1 min
Transfer the column to clean 1.5 mL tube, add 30µL elution buffer directly to column. Let stand for 1 min
Centrifuge 30 seconds to elute DNA

July 18, 2018

Transformation with RbCl JM109 Cells

Liz, Peter

**See Protocol from 7/16/18**

Transforming failed transformations from 7/17 + more:

BBa_J04450 (RFP), BBa_K1316012 (Mtr + T7 Lac O.) from 2017 and 2018 kit, TesA (from ligation + now purified w/ PCR purif), FadK (from ligation + now purified w/ PCR purif), FadD (from ligation + now purified w/ PCR purif), Blank

Notes/Deviations:

Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube (only one tube for the blank)
Heat-shocked for 30 seconds based on results from transformations of ccmA-H (done on 7/16)
Pooled like tubes (same vector) after 1 hour incubation

Results (7/19/18):

Part/ Vector	Transformation Result
BBa_J04450 (RFP)	1 colony (O.C. on 7/19)
BBa_K1316012 (Mtr + T7 Lac O.) 2017 kit	No growth
BBa_K1316012 (Mtr + T7 Lac O.) 2018 kit	No growth
TesA (from ligation + now purified w/ PCR purif)	1 colony (O.C. on 7/19)
FadK (from ligation + now purified w/ PCR purif)	1 colony (O.C. on 7/19)
FadK (from ligation + now purified w/ PCR purif)	No growth
FadD (from ligation + now purified w/ PCR purif)	No growth
Blank	No growth

July 18, 2018

LB + Chloramphenicol Plates Preparation

Ryan

Preparing Chloramphenicol LB plates (24)

Collected and measured out ingredients for LB media and added to bottle
Q/s’d with dH₂0 to 400 and shook bottle to dissolve ingredients
Stirred in beaker for 30 min
Poured into grad cylinder to finish Q/s to 500 mL
Poured back into bottle and stirred for ~ a minute
Autoclaved on S03 setting for 30 minutes (autoclaving less than a liter of liquid)
Added 0.5 mL of chloramphenicol (1000x, 35 mg/mL) to solution
Put them in the hood with the plates half open w/ agar side on the bottom to get rid of condensation

July 19, 2018

Miniprep and glycerol stocks of cymA ligation and B0015

Shannon

Used Zyppy Miniprep Kit and instructions from the manufacturer’s website
Add 600µL of each bacterial culture to separate 1.5mL tubes
Add 100µL 7x lysis buffer, mix by inverting
Add 350µL cold neutralization buffer (yellow), mix by inverting
Centrifuge at 13,000g for 3 min
Transfer supernatant (about 900µL) into column, place column in collection tube
Centrifuge for 15 seconds
Discard flow-through, put column back into the same collection tube
Add 200µL endo wash buffer, centrifuge 20 seconds
Add 400µL Zippy wash buffer to column. Centrifuge 1 min
Transfer the column to clean 1.5 mL tube, add 30µL elution buffer directly to column. Let stand for 1 min
Centrifuge 30 seconds to elute DNA
Made glycerol stocks by combining 0.75mL of 40% glycerol and 0.75mL overnight culture, stored at -80C

July 19, 2018

PCR Purification of fadD and fadK

Shannon

Using Lambda Biotech Column Pure PCR Cleanup Kit
Mix 20µL PCR reaction with 100µL binding buffer
Load onto spin column, centrifuge at full speed (16,100g) for 1 min
Discard flow-through
Add 700µL wash buffer, centrifuge 1 min
Discard flow-through, centrifuge again 1 min
Transfer column to clean 1.5mL tube
Add 30µL elution buffer, let sit 1 min, centrifuge 1 min to elute DNA

July 19, 2018

Restriction Digest of CymA and B0015

Peter

B0015 (Terminator):

Component	Amount
PstI (HF)	1µL
CutSmart	1µL (should have done 2µL)
DNA template	100ng x 1µL/53.8ng = 1.86µL
Water	16.14µL (To 20µL)

Order: water, buffer, DNA, enzymes
Spin down in tabletop centrifuge
Heat blocked at 37C for 10 min
Heat-killed on block at 80C for 15 min

CymA:

Component	Amount
PstI (HF)	1µL
CutSmart	1µL (should have done 2µL)
DNA template	5µL (20 ng/µL) -wanted a lot of it)
Water	13 µL (To 20µL)

Order: water, buffer, DNA, enzymes
Spin down in tabletop centrifuge
Heat blocked at 37C for 10 min
Heat-killed on block at 80C for 15 min

MtrCAB (2017 or 2018?):

Component	Amount
PstI (HF)	1µL
CutSmart	1µL (should have done 2µL)
DNA template	100ng x 1µL/112.5ng = 0.89µL
Water	17.11 µL (To 20µL)

Order: water, buffer, DNA, enzymes
Spin down in tabletop centrifuge
Heat blocked at 37C for 10 min
Heat-killed on block at 80C for 15 min

July 19, 2018

PCR Purification of phz 1F-3R Gel Extraction PCR and a Gel

Sricharan

PCR purified product from yesterday (phz 1F-3R gel extraction PCR)
Used Lambda Biotech Kit protocol, eluted with 30µL buffer

Agarose gel (1%, large 35mL)

Lane	1	2	3	4	5	6	7	8	9	10
Item	2-log ladder	phz 1F-3R Gel Extraction PCR	cymA MP	B0015 MP	K1316012 plasmid (from 2017 Kit)	fadD PCR2 with correct annealing temp	fadD PCR0 diluted to 40ng/30µL	fadK PCR2 with correct annealing temp	fadK PCR0 diluted to 40ng/30µL	2-log ladder
Amt (µL)	5	3+2 dH2O+1 dye	5+1 dye	5+1 dye	5+1 dye	5+1 dye	5+1 dye	5+1 dye	5+1 dye	6

Note that lanes 3-5 are digested with PstI

Seems that fadD PCR of the coding region (“PCR2”) is successful since it is significantly amplified past that of template.
Need to redo fadK PCR2
Also cymA and B0015 might be around the same size, but hard to tell here, need to retry the gel

July 19, 2018

Overnight Cultures of TesA and J04450 (RFP)

Peter

Overnight Cultures:

TesA
BBa_J04450 (RFP)

Results (7/20/18):

They worked: proceed to miniprep and glycerol stocking

July 20, 2018

PCR2 of FadK and PCR of ‘TesA(L109P) Basic Part from miniprep (7/18/18)

Peter, Sricharan

FadK PCR2:

Set thermocycler:

Phase	Time (sec)	Temp. (C)
Initial denaturation	30	98
35 cycles	10	98
	30	72 (from NEB calculator)
	48	72
Final extension	10 minutes	72
Hold	∞	4

Add to tubes in this order:

Component	Amount (µL)
dH₂O	12.11
HF buffer	4
dNTP mix	0.4
10mM forward primer (bad)	1
10mM reverse primer (bad)	1
DMSO	0.6
Template DNA (FadK PCR0)	0.69 (10 ng)
phusion	0.2

All components were kept on ice

‘TesA (L109P):

Set thermocycler:

Phase	Time (sec)	Temp. (C)
Initial denaturation	30	98
35 cycles	10	98
	30	72 (from NEB calculator)
	26	72
Final extension	10 minutes	72
Hold	∞	4

Add to tubes in this order:

Component	Amount (µL)
dH₂O	12.54
HF buffer	4
dNTP mix	0.4
10mM forward primer (bad)	1
10mM reverse primer (bad)	1
DMSO	0.6
Template DNA (‘TesA L109P from miniprep)	0.26 (10 ng)
phusion	0.2

All components were kept on ice

July 20, 2018

Gel of FadK PCR2, ‘TesA (L109P) basic part, and other stuff (see Sricharan’s notebook)

Peter, Ryan

Wells:

1	2	3	4	5	6
2 Log Ladder	fadK PCR2 HF	fadK PCR0 diluted to 0.69µL/30µL	fadK PCR2 GC	‘tesA(L109P) PCR Basic	K1316012 (mtrCAB) 2017 Kit Digest w/ PstI

7	8	9	10	11	12
2 Log Ladder	‘tesA(L109P) Miniprep Digest w/PstI	Kit 7, Well 2H 2018 Digest w/PstI	J04450 Miniprep Digest 3x	See lane 10	See Lane 1

Note that 6µL of ladder added to lanes 1, 7, 12; 5µL of item and 1µL of loading dye added to all other lanes

Results:

1	2	3	4	5	6

7	8	9	10	11	12

July 20, 2018

Restriction 5 things

Peter

Digest of J04450 (RFP), ‘TesA (L109P), FadD PCR2, Mtr 2016 (K1316012), Kit 7 Well 2H

J04450 (RFP):

Component	Amount
XBaI	1µL
SpeI	1µL
DpNI	1µL
CutSmart	1µL (should have done 2µL)
DNA template	10µL
dH₂O	6µL (To 20µL)

Order: water, buffer, DNA, enzymes
Spin down in tabletop centrifuge
Heat blocked at 37C for 10 min
Heat-killed on block at 80C for 15 min

CymA:

Component	Amount
PstI (HF)	1µL
CutSmart	1µL (should have done 2µL)
DNA template	5µL (20 ng/µL) -wanted a lot of it)
Water	13 µL (To 20µL)

Order: water, buffer, DNA, enzymes
Spin down in tabletop centrifuge
Heat blocked at 37C for 10 min
Heat-killed on block at 80C for 15 min

MtrCAB (2017 or 2018?):

Component	Amount
PstI (HF)	1µL
CutSmart	1µL (should have done 2µL)
DNA template	100ng x 1µL/112.5ng = 0.89µL
Water	17.11 µL (To 20µL)

Order: water, buffer, DNA, enzymes
Spin down in tabletop centrifuge
Heat blocked at 37C for 10 min
Heat-killed on block at 80C for 15 min

July 23, 2018

LB + Cam, LB + Kan Plates Preparation

Ryan

Preparing Chloramphenicol LB plates (18):

Collected and measured out ingredients for LB media and added to bottle
Q/s’d with dH20 to 350 mL and shook bottle to dissolve ingredients
Stirred in beaker for 30 min
Poured into grad cylinder to finish Q/s to 375 mL
Poured back into bottle and stirred for ~ a minute
Autoclaved on S03 setting for 30 minutes (autoclaving less than a liter of liquid)
Split solution in half such that 9 plates are for Cam and 9 plates are for Kan
Added 0.1875 mL of chloramphenicol (1000x, 35 mg/mL) to solution in one half, 0.1875 mL of kanamycin (1000x, 35 mg/mL) to other
Let plates cool for an hour on lab bench

July 23, 2018

Digest of J04450 and psb1C3

Peter and Shannon

Add reagents in order listed
Centrifuge briefly
Heat block at 37C (45 min for psb1C3, 10 min for the rest)
Heat block at 80C for 15 min to heat kill enzymes

psb1C3, I718008, K1433005, K1316012

Component	Amount (µL)
Water (milliQ)	psb1C3: 12
	1718008: 14.83
	K1433005: 14.14
	K1316012: 15
CutSmart	2
DNA	psb1C3: 4
	1718008: 1.17
	K1433005: 1.86
	K1316012:1
EcoRI	1
PstI	1

J04450

Component	Amount (µL)
Water (milliQ)	15.65
CutSmart	2
DNA	0.35
SpaI-HF	1
XbaI	1

July 23, 2018

Reviving Glycerol Stocks

Shannon

Put glycerol stocks on ice (try not to let them thaw too much)
Stab into the glycerol stock with a p20 pipette tip
Use pipette tip to t-streak on a place with the correct antibiotic
Incubate overnight at 37C
Revived: J23114, J23110, J23100, and cymA. Was going to revive K864400 but could not find it in the -80C freezer
Note: There were three tubes labelled J23110, and one tube that had the label wiped off. These may have been mislabelled. I believe two are J23110 and two are J23100. The two that have more color should be the stronger promoter, and the two with less color should be the weaker promoter. I labelled one plate “S” and one “W” for strong and weak.
Note: For the tube labelled J23110, I don’t know which of the three “J23110” tubes I used. I did this one before realizing the tubes may have been mislabelled.

July 23, 2018

Agarose Gel for Extraction and Checking stuff

Sricharan

Another 1% agarose gel
Liz gel extracted lanes 2 and 3 at pSB1C3 band (~2kb)

1	2	3	4	5	6
2-log ladder	J04450 Digest	J04450 Digest	K143305 Digest 7/23	I718008 Digest 7/23	K14316012 Digest 7/23

Note that 6µL of ladder to lane 1; 5µL item and 1µL dye to every other well. Lanes 2 - 5 are sourced from minipreps. Lane 6 is just DNA from the distribution kit we wanted to test.

July 23, 2018

Gel Extraction of J04450 3x Digest

Shannon

Excise DNA from gel, add to 1.5mL tube. 184mg were excised.
Add 3x volume ADB. 555mL were added.
Incubate at 45C for 5-10 min until dissolved
Transfer to spin column
Centrifuge 60 sec at 10,000g. Discard flow-through.
Add 200µL wash buffer, centrifuge 30 sec. Discard flow through. Repeat.
Add 10µL elution buffer directly onto column. Let sit for 1 min.
Place column in clean 1.5mL tube. Centrifuge 60 sec.
Store at -20C.
Note: the elution buffer was from the invitrogen kit.

July 23, 2018

Gel Extraction of J04450 3x Digest

Shannon

Excise DNA from gel, add to 1.5mL tube. 184mg were excised.
Add 3x volume ADB. 555mL were added.
Incubate at 45C for 5-10 min until dissolved
Transfer to spin column
Centrifuge 60 sec at 10,000g. Discard flow-through.
Add 200µL wash buffer, centrifuge 30 sec. Discard flow through. Repeat.
Add 10µL elution buffer directly onto column. Let sit for 1 min.
Place column in clean 1.5mL tube. Centrifuge 60 sec.
Store at -20C.
Note: the elution buffer was from the invitrogen kit.

July 23, 2018

J04450 Digest

Liz

Set heat blocks to 37C and 80C

Components (in order)	Amount (µL)
milliQ water	6
Cutsmart	2
DNA (J04450 miniprep SK 7/20, 287ng/µL)	10
SpeI-HF	1
XbaI	1

Incubated at 37C for 10 min
Heat shocked at 15 min

July 24, 2018

PCR of PSB1C3 Template from RFP mini-prep

Peter, Sricharan

Set thermocycler:

Phase	Time (sec)	Temp. (C)
Initial denaturation	30	98
35 cycles	10	98
	30	71 (from NEB calculator)
	60	72
Final extension	10 minutes	72
Hold	∞	4

*set to 50 µL reaction

Add to tubes in this order:

Component	Amount (µL)
dH₂O	31.65
HF buffer	10
dNTP mix	1
10mM forward primer (bad)	2.5
10mM reverse primer (bad)	2.5
DMSO	1.5
Template DNA (FadK PCR0)	0.348 (5 ng - from 10x dilute of miniprep)
phusion	0.2

All components were kept on ice
Used lambda PCR cleanup kit
Nano-dropped @ 48.8 ng/µL
Did gel extract for linearized plasmid for Gibsons (no longer using for Gibsons)

July 24, 2018

Overnight Cultures

Shannon

Did overnight cultures of J23114, J23110, J23100, and cymA
Combine 5mL LB and 5µL antibiotic (ampicilin for J23114, J23110, and J23100 and chloramphenicol for cymA)
Put in shaker at 4:20pm
Note: “S” and “W” are either J23110 or J23100. “S” should be the stronger promoter, “w” should be the weaker promoter.
Note: The tube labelled “J23110” may be J23100. Check color against “S” and “W”?

July 24, 2018

Digest of psB1C3, acr 1, acr 3, fadD, fadK, B0015, cymA, pSB1K3, ccmA-H

Ryan and Liz

Add reagents in order listed
Centrifuge briefly
Heat block at 37C (45 min for psb1C3, 10 min for the rest)
Heat block at 80C for 15 min to heat kill enzymes

fadD, fadK, cymA, pSB1K3

Component	Amount (µL)
Water (milliQ)	fadD: 11.7
	fadD: 9
	cymA: 11
	pSB1K3: 12
CutSmart	2
DNA	fadD: 4.3
	fadK: 7
	cymA: 5
	pSB1K3: 4
EcoRI	1
Pstl	1

pSB1C3

Component	Amount (µL)
CutSmart	.5
DNA	4
EcoRI	.25
PstI	.25

Acr 1

Component	Amount (µL)
CutSmart	1.25
DNA	10
Pstl	1.25

Acr 3

Component	Amount (µL)
CutSmart	1.25
DNA	10
Pstl	1.25

B0015

Component	Amount (µL)
Water (milliQ)	14.1
CutSmart	2
DNA	1.86
Xbal	1
Pst1	1

ccmA-H

Component	Amount (µL)
Water (milliQ)	15.4
CutSmart	2
DNA	0.57
EcoRI	1
Spel	1

July 24, 2018

Gibson Reactons, Circular Version

Sricharan

Planning Gibson reactions to assemble acr. We realized the overlaps between our acr inserts and the pSB1C3 backbone isn’t great. We decided on a couple reactions to see if we can use these inserts

For 50ng pSB1C3, or 0.037pmol of backbone, using the NEB kit, we want about 2x excess of inserts. Using NEB calculator to figure out mass of insert required:

acr Synthesis 1	501bp	26ng
acr Synthesis 2	529bp	27.5ng
acr Synthesis 3	642bp	33.4ng

Gibson 1: Negative Control Reaction

Item	Amount (µL)
pSB1C3 from J04450 (XbaI and SpeI digested)	1.43
dH₂O	9.41
NEB Gibson Master Mix	10.8

Gibson 2: acr Assembly

To have adequate overlap between acr inserts and pSB1C3, we’d need to digest something with pSB1C3 backbone with XbaI and SpeI (poorly designed inserts on our part). We did this to J04450 and gel extracted this band. However, this means that there’s a risk of a) contamination with J04450 plasmid and b) religation of the sticky ends of XbaI and SpeI. That’s why we’re running the negative control above and are considering reordering some gBlocks to make Gibsons go smoother. Other option would be to get new primers to amplify pSB1C3 with the overlaps designed into the primers.

Item	Concentration (ng/µL)	Amount (µL)
pSB1C3 from J04450 (XbaI and SpeI digested)	35	1.43
acr Synthesis 1 (EcoRI digested)	100/12.5	3.01
acr Synthesis 2	10	2.54
acr Synthesis 3 (PstI digested)	100/12.5	3.86
NEB Gibson Master Mix		10.8

Gibson 3: acr Assembly Alternate

Strategy - ligating pSB1C3 cut with EcoRI and PstI, acr Synthesis 1 cut with EcoRI, acr Synthesis 3 cut with PstI
Then this ligation is added to the Gibson reaction with acr Synthesis 2
Back calculated approx amts of each component required so that concentrations of digest/ligation is high as possible

For 70ng digested pSB1C3, or 0.052 pmol, a 1:1 excess of insert means for the ligation:

Item	Concentration (ng/µL)	Amount (µL)
pSB1C3 Linear Backbone (EcoRI and PstI digested)	20	3.5
acr Synthesis 1 (EcoRI digested)	100/12.5	2.12
acr Synthesis 3 (PstI digested)	100/12.5	2.12
T4 Ligase	100/12.5	0.46
T4 Ligation Buffer		0.91

(Coordinated on digest concentrations with Liz, used NEB calculator to obtain ng amounts of each insert)
Ligation was incubated at 10 min, RT; Heat killed at 65 °C for 20 min
Added 7µL of this ligation (0.04pmol, theoretical max of 78ng ligated product) to 3µL of acr Synthesis 2 (0.088pmol or 30ng)

July 25, 2018

Gibson Reactons, Circular Version, Day 2

Sricharan

Finished Gibson 2’ (same as 2 but redid since we were unsure about digests), 3 as described yesterday
Next day, got only 1 colony from all Gibsons, from Strategy 2 … later realized this was probably religated plasmid

July 25, 2018

Miniprep of J23114, “J23110”, “S”, “W”, and cymA

Shannon

“J23110” could be J23110 or J23100. “S” should be J23110, and “W” should be J23100.
Spin down 1.5mL of overnight culture, discard supernatant, add another 1.5mL, spin down, discard supernatant, and rehydrate the pellet in 600µL dH₂O.

Spun at 16,100g for 1 min each time

Add 100µL lysis buffer, invert to mix
Add 200µL cold neutralization buffer (kept on ice), invert to mix
Centrifuge at 13,000 for 3 min
Transfer supernatant to column (about 900µL)
Place column in collection tube. Centrifuge 15 sec.
Discard flow-through, put back into collection tube
Add 200µL endo-wash buffer, centrifuge 30 sec.
Add 400µL wash buffer, centrifuge 1 min
Transfer column to clean 1.5mL tube. Add 30µL elution buffer. Let stand 1 min.
Centrifuge 30 sec to elute DNA

July 25, 2018

Transformation with RbCl JM109 Cells

Sricharan and Peter

**See Protocol from 7/16/18**

Transforming:

BBa_K1316012 (Mtr + T7 Lac O.) from 2016 kit w/ Strataclone Commercial Cells and our JM109 cells, FadD (from ligation + now purified w/ PCR purif), FadK (from ligation + now purified w/ PCR purif), BBa_J23100 (Strong Anderson Promoter), BBa_J23110 (Medium Anderson Promoter), BBa_K864400 (pTac promoter), ACR Gibson 2 (7/24), Gibson control (7/24), ccmA-H 3A (7/24), ACR Gibson 2’ (7/25), ACR Gibson 3 (7/25)

Notes/Deviations

Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube (Gibsons each get 1 μL per tube instead of 0.5, ligations should have maybe gotten 1 μL per tube instead of 0.5, 1 μL for both Mtr tubes of different cells)
Took DNA from diluted Gibson solutions
Heat-shocked for 30 seconds based on results from transformations of ccmA-H (done on 7/16)
Pooled like tubes (same vector) after 1 hour incubation
Amp plates for Anderson Promoters and Kanamycin plate for ccmA-H 3A

Results (7/26/18):

Part/ Vector	Transformation Result
BBa_K1316012 (Mtr + T7 Lac O.) 2016 kit Strataclone	1 colony (O.C. on 7/26)
BBa_K1316012 (Mtr + T7 Lac O.) 2018 kit Our JM109 Cells	No growth
FadD (from ligation + now purified w/ PCR purif)	No growth
FadK (from ligation + now purified w/ PCR purif)	1 colony (O.C. on 7/26)
BBa_J23100 (Anderson Strong)	Good growth (O.C. on 7/26)
BBa_J23110 (Anderson Medium)	Good growth (O.C. on 7/26)
BBa_K864400 (pTac)	No growth
ACR Gibson 2 (7/24)	1 colony - could be plasmid backbone ligated to itself (O.C. on 7/26)
Gibson Control (7/24)	1 colony - shows that plasmid backbone can ligate to itself (O.C. on 7/26)
ccmA-H 3A (7/24)	Good growth (O.C. on 7/26)
ACR Gibson 2’ (7/25)	No growth
ACR Gibson 3 (7/25)	No growth? (Might not have carried out this transformation)

July 26, 2018

Resuspension of phz G-Blocks (end and 3) for Gibson

Peter

Centrifuge tube for 3-5 seconds on table-top centrifuge
Add TE to reach a final concentration of 10 ng/µL

Phz end 500ng / 50µL = 10 ng/µL
Phz 3 250ng/ 25µL = 10 ng/µL

Vortex briefly
Incubate @ 50 C for 20 minutes
Vortex and briefly centrifuge
Store in -20 C

July 26, 2018

Diagnostic Gel of B0015 and CymA and Gel Extract of PSB1C3

Peter and Liz

Wells:

1	2	3	4	5
2 Log Ladder	CymA	B0015	PSB1C3 Extract	PSB1C3 Extract
-6 µL ladder	-5 µL Double Digest -1 µL Loading Dye	-5 µL Double Digest -1 µL Loading Dye	-5 µL Digest -1 µL Loading Dye	-5 µL Digest -1 µL Loading Dye

Gel Extract (Zyppy gel extraction kit):

Extracted last 2 wells of gel above to get pSB1C3 (brightest band)

Other DNA in the band is RFP and ‘tesA (length of ‘tesA is questionable but we don’t really care about that since we just want backbone)

Pice of gel extracted: 0.1268 = 126.8mg → added 126.8 x 3 = 380.4µL ADP
Incubated at 42C in heat block until agarose dissolved
Put into spin column and centrifuged 1 min at 16,100g then pipetted out flow-through
Added 200µL wash buffer and centrifuged 30s at 16,100g (did this step 2x)
Added 6µL elution buffer (from invitrogen PCR purification kit), let sit for 1 min, then centrifuged 1 min at 16,100g

July 26, 2018

3A Assembly of cymA and Anderson promoters into psb1K3

Shannon

Need a 1:1 molarity of backbone to inserts, so use 8.768ng cymA and 0.397ng J23110
Dilute promoter solutions

We need 0.397ng of each. If undiluted, you would need to small an amount to pipette
Add 1µL of the J23100/J23110 digestion (concentration = 57.48 ng/µL and 57.81ng/µL) to 29µL dH2O (new concentration = 1.916ng/µL and 1.927ng/µL).
Add 1µL of the J23114 digestion (concentration = 469.18ng/µL) to 249µL dH2O (new concentration = 1.88ng/µL)

Component	Amount (µL)
Backbone (psb1K3)	5
cymA	1.75
Promoter	0.2
T4 ligase buffer	2.5
T4 enzyme	1.25
dH₂O	14.3

Combine components in order listed
Ligate at 16C for 30min
Heat kill at 80 C for 20 min
Note: accidently put 1.2µL of J23100 into that reaction.
Note: I also forgot to dilute the promoters. Will have to re-do this tomorrow.

July 26, 2018

Some Planning

Sricharan

Planned out a Gibson for phz, but realized we don’t have enough of 1F-3R and 4F-6R fragments PCRed
Toni will run PCR of above two fragments again

July 26, 2018

Agarose Gel of Minipreps & PCR of Gibson 1F-3R and 4F-6R

Sricharan

Lane	1	2	3	4	5	6
Item	fadK MP Digest	ccmA-H + B0015 MP Digest	J23110 MP Digest	J23100 MP Digest	Phz Gibson 2 MP Digest	K1316012 MP Digest

7	8	9	10	11	12
Ladder (2-log)	Gibson Ctrl MP Digest	pSB1C3 PCR Digest	phz 1F-3R Gibson PCR	phz 4F-6R Gibson PCR	Nothing

All items digested w/PSTI except pSB1C3 (w/ EcoRI, PstI, DpnI - concentrations are off so might not work)

5µL item, 1µL loading dye, Dated 7/27/2018

July 26, 2018

Restriction Digests for phz Gibson and CymA 3A

Liz

Start of phz: (adjusted so no water for higher concentration)

Component	Amount (µL)
Water	0
CutSmart	1.25
DNA (rehydrated start of phz gBlock)	10 (100ng x 1µL/10ng = 10µL → 10/4 = 2.5µL enzymes/water)
EcoRI	1.25

End of phz: (adjusted so no water for higher concentration)

Component	Amount (µL)
Water	0
CutSmart	1.25
DNA (rehydrated start of phz gBlock)	10 (100ng x 1µL/10ng = 10µL → 10/4 = 2.5µL enzymes/water)
EcoRI	1.25

Medium Anderson Promoter (J23110):

Component	Amount (µL)
Water	14.27
CutSmart	2
DNA (J23110 miniprep)	1.73 (100ng x 1µL/57.81ng = 1.73µL)
EcoRI	1
Spel	1

cymA:

Component	Amount (µL)
Water	15.27
CutSmart	2
DNA (cymA miniprep)	0.83 (100ng x 1µL/120.6ng = 0.83µL)
XbaI	1
PstI	1

Strong Anderson Promoter (J23100):

Component	Amount (µL)
Water	14.26
CutSmart	2
DNA (J23100 miniprep)	1.74 (100ng x 1µL/57.48ng = 1.74µL)
EcoRI	1
Spel	1

Weak Anderson Promoter (J23114):

Component	Amount (µL)
Water	15.79
CutSmart	2
DNA (rehydrated start of phz gBlock)	0.213 (100ng x 1µL/469.18ng = 0.213µL)
EcoRI	1
Spel	1

RFP miniprep (J04450):

Component	Amount (µL)
Water	6
CutSmart	2
DNA	10
XbaI	1
Spel	1

For DNA used all of remaining miniprep and then another random miniprep (‘tesA?) to try to get to 10µL to maximize backbone
Heat blocked at 37C for 1 hr then 80C for 15 min (put in thermocycler at 5µL reaction)

July 26, 2018

Transformations

Liz

Used 2 tubes of RbCl JM109 cells for each sample

Gibson control (1µL DNA per tube)
acr gibson 2 (1µL DNA per tube)
acr gibson 2’ (1µL DNA per tube)
K864400 (0.5µL DNA per tube)
fadD ligation (1µL DNA per tube)

Followed transformation protocol (7/16/2018)

July 27, 2018

Restriction Digests

Shannon

FadK, J23110, J23100, gibson 2 transformation, gibson control

Component	Amount (µL)
Water (milliQ)	For fadK: 16.57
	For J23110:16.26
	For J23100: 16.80
	For gibson 2 transformation: 16.56
	For gibson control: 16.54
CutSmart	2
DNA	For fadK: 0.43
	For J23110: 0.74
	For J23100: 0.20
	For gibson 2 transformation: 0.44
	For gibson control: 0.46
Pstl	1

ccmA-H + B0015

Component	Amount (µL)
Water (milliQ)	14.97
CutSmart	2
DNA	1.03
PstI	1
XbaI	1

Stratoclone K1316012

Component	Amount (µL)
Water (milliQ)	15.79
CutSmart	2
DNA	0.21
PstI	1
BspHI	1

psb1K3

Component	Amount (µL)
Water (milliQ)	12
CutSmart	2
DNA	4
PstI	1
DpnI	1

psb1C3

Component	Amount (µL)
Water (milliQ)	24.4
CutSmart	9.76
DNA	10
EcoRI	4.88
PstI	4.88
DpnI	4.88

Add reagents in order listed
Centrifuge briefly
Heat block at 37C

10 min for all except psb1K3 and psb1C3. 45 min for those.

Heat block at 80C for 15 min.
Note: shouldn’t have increased the volume for the components in psb1C3. Did not need to scale.

August 2, 2018

Resuspending G-Blocks

Shannon

Resuspended phz start, phz end, acr1, acr3, pmt1231.1, pmt1231.2
500ng of each g-block was deliverd, so 50µL TE was added to each for a final concentration of 10ng/µL.
Centrifuge tubes for 3-5 sec on tabletop centrifuge
Add TE
Vortex briefly
Incubate at 50C for 20min
Briefly vortex and cetrifuge
Store at -20C

July 27, 2018

Making SOC Medium

Peter and Ryan

Bottle 1: Will be SOC Medium
Bottle 2: 2 M MgCl₂
Bottle 3: 1 M Glucose

Add to Bottle 1:

Component	Amount
Water (dH₂O)	950 mL
Bacto-tryptone	20 g
Bacto-yeast extract	5 g
NaCl	0.5 g
KCl	0.1863775 g 10 ml (0.01 L) of 250 mM solution (0.25 M solution) 0.01 L x 0.25mol/1L x 74.551g/1mol = 0.1863775g

Shake and used stir bar to dissolve components
pH to 7.0 w/ 5 N NaOH (~ 0.2 ml) -- didn’t have any so used 1 N NaOH
Adjust volume to 1 liter w/ dH₂O (mark 1 liter point on bottle before adding components)
Autoclave with Bottle 2

Add to Bottle 2:

Component	Amount
Water (dH₂O)	90mL
MgCl₂	19 g

After components are dissolved, adjust volume to 100 mL w/ dH₂O (mark 100 mL point on bottle before adding components)
Autoclave with Bottle 1
Add 5 mL to Bottle 1 AFTER AUTOCLAVING

Bottle 3:

Component	Amount
Water (dH₂O)	90mL
Glucose	18g

After Glucose is dissolved, adjust volume to 100 mL w/ dH2O (mark 100 mL point on bottle before adding components)
Sterilize by filtration through 0.22-micron filter
Add 20 mL to Bottle 1 AFTER AUTOCLAVING

July 27, 2018

Minipreps

Liz

Miniprepped overnight cultures using zyppy miniprep kit

fadK ligation
ccmA-H + B0015 3A
J23110
J23100
Gibson acr 2 transformation
Gibson control
Strataclone K1316012

Used 3mL each overnight, spun down, and rehydrated in 600µL water
Eluted in 30µL elution buffer

July 30, 2018

3 Digests and a Ligation (And a gel)

Sricharan and Liz

Digesting 13µL of mtrCAB (K1316012) with BspHI and PstI to be ligated to start and assembled to pSB1K3
Digesting 15µL of pSB1C3 (Peter’s PCR) and pSB1K3 (Linearized backbones kit) with EcoRI and PstI and DpnI (ligations in the future)
14µL ccmA-H + B0015 diagnostic digest with EcoRI to check on a gel
6µL of Mtr Start (gBlock) digested with EcoRI and BspHI to be ligated to rest of mtrCAB and assembled to
13µL of ccmA-H (K917006) also digested with EcoRI and SpeI for future 3A assembly
Using standard digestion protocol (all 20µL digests)

Ligation:
mtrCAB (TU Delft part) + synthesized mtr start (rehydrated gBlock) + pSB1K3

Component	Amount (µL)
water	3.24
T4 ligase buffer	0.67
Backbone (pSB1K3 from digestion above)	1.33 (25ng x 20µL/375ng = 1.33)
Digested mtr start (equimolar to backbone)	0.28 (1.418ng x 20µL/100ng = 0.28)
Digested mtrCAB (equimolar to backbone)	0.82 (58.87ng x 20µL/1437.36ng = 0.82)
T4 ligase	0.33

Scaled to 6.67µL reaction based on 25ng plasmid backbone

Lane	1	2	3
Item	2-log ladder	ccmA-H + B0015 cut with EcoRI (7/30), 5µL	pSB1C3 cut w/ EcoRI, PstI, DpnI (7/30), 1µL

July 30, 2018

Transformation with RbCl JM109 Cells

Sricharan and Peter

**See Protocol from 7/16/18**

Transforming:

FadD (from ligation + now purified w/ PCR purif), mtrCAB 3A (mtrCAB + New Start + PSBIC3), BBa_K864400 (pTac promoter)

Notes/Deviations:

Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube.
Ligation gets1 μL per tube instead of 0.5
Took DNA from diluted Gibson solutions
Heat-shocked for 30 seconds
Pooled like tubes (same vector) after 1 hour incubation
Kanamycin plate for mtrCAB 3A new start

Results (7/31/18):

Part/Vector	Transformation Result
FadD (from ligation + now purified w/ PCR purif)	No growth
mtrCAB 3A (mtrCAB + New Start + PSBIC3)	Good growth (O.C. on 7/31)
BBa_K864400 (pTac)	Good growth (O.C. on 7/31)

July 31, 2018

PCR of phz 4f-6R, ‘tesA (L109P), Basic cymA

Peter

Notes:

phz 4F-6R: 2.588 kb
Basic ‘tesA (L109P): 0.57 kb
Basic cymA: 0.595 kb

Set thermocycler:

Phase	Time (sec)	Temp. (C)
Initial denaturation	30	98
35 cycles	10	98
	30	Phz: 67
	Phz: 77.6s	‘tesA & CymA: 72 (from NEB calculator)
	‘tesA & CymA: 17.3	72
Final Extension	10 minutes	72
Hold	∞	4

*set to 50 µL reaction for phz 4F-6R
*set to 20 µL reaction for ‘tesA and cymA

Phz 4F-6R Tube:

Component	Amount (µL)
dH₂O	29
HF buffer	10
dNTP mix	1
10mM forward primer (bad)	2.5
10mM reverse primer (bad)	2.5
DMSO	1.5
Template DNA (Gibson of 4F-6R)	3 (10ng x 1µL/3.785ng = 2.642µL) -rounding up to 3 µL
phusion	0.5

‘tesA (L109P):

Component	Amount (µL)
dH₂O	12.6
HF buffer	4
dNTP mix	0.4
10mM forward primer (bad)	1
10mM reverse primer (bad)	1
DMSO	0.6
Template DNA	0.211 (10ng x 1µL/47.5ng = 0.211µL) -from 4x dilute of miniprep
phusion	0.2

Cyma (Basic) Tube:

Component	Amount (µL)
dH₂O	12.6
HF buffer	4
dNTP mix	0.4
10mM forward primer (bad)	1
10mM reverse primer (bad)	1
DMSO	0.6
Template DNA (FadK PCR0)	0.249 (10ng x 1µL/120.6ng = 0.249µL) -from 3x dilute of miniprep
phusion	0.2

All components were kept on ice
Used lambda PCR cleanup kit

August 1, 2018

Gel

Peter

Lane	1	2	3	4	5	6	7	8
Item	K86440 0(pTac)	Basic CymA (pcr purif.)	Basic ‘tesA (L109P)	2-log ladder	Failed Lane	Failed Lane	phz 4F-6R (pcr purif.)	mtrCAB new st. 3A

August 1, 2018

So Many Things

Sricharan

Miniprepping Peter’s OCs of MtrCAB (3A Assembled w/ new start) ->3 colonies picked, 3 OCs
Minipreps done with Zymo Plasmid MP Kit
BBa_K864400 (pTac) is 1 OC

250µL of each on ice, ready to add glycerol once we know which ones are good size… added glycerol

Lots of Digests (all in CutSmart buffer, all volumes in µL):

Sample	mtrCAB new st (1-3)	K864400 (4)	ccmA-H (5)	fadK (6)	cymA Basic (7)	‘tesA Basic (8)
dH₂0	0	0	11	11	0	0
Buffer	2	2	2	2	2	2
DNA	16	16	5	5	16	16
Enzyme	1 XbaI 1 PstI	1 EcoRI 1 SpeI	1 EcoRI 1 SpeI	1 XbaI 1 PstI	1 EcoRI 1 PstI	1 EcoRI 1 PstI

1-4 by PC, 5-8 by SK

3A Assemblies … Using NEB Ligation Calculator for amounts, 1:1:1 molar ratios

1) Anderson (J23110) + cymA + pSB1K3

Component	Mass (ng)	Volume (µL)	Notes
pSB1K3 (SK 7/30)	25	1.33	Digested 7/30
J23110 Digest (LCC 7/26)	33.84	6.77	2983 bp
cymA Digest (LCC 7/26)	32.25	6.45	2843 bp
T4 Ligase Buffer	-	2	-
T4 Ligase	-	1	-
dH₂O	-	2.45	-

2) Anderson (J23110) + mtrCAB w/new st + pSB1C3

Component	Mass (ng)	Volume (µL)	Notes
pSB1C3 (SK 7/30)	25	0.68	Digested 7/30
J23110 Digest (LCC 7/26)	36.03	7.21	-
mtrCAB w/new st Digest (8/1)	89.23	2.85	> 388 bp
T4 Ligase Buffer	-	2	-
T4 Ligase	-	1	-
dH₂O	-	6.26	-

3) ccmA-H (K917006) + B0015 + pSB1K3 … 10µL rxn

Component	Mass (ng)	Volume (µL)	Notes
pSB1K3 (SK 7/30)	25	1.33	2204 bp
B0015 Digest (LCC 7/24)	24.94	4.99	2199 bp
ccmA-H Digest (SK 8/1)	96.84	2.2	8537 bp
T4 Ligase Buffer	-	1	-
T4 Ligase	-	0.5	-
dH₂O	-	0	-

pTac (K864400) + fadK + pSB1K3 … 10µL rxn

Component	Mass (ng)	Volume (µL)	Notes
pSB1K3 (SK 7/30)	25	1.33	2204 bp
pTac Digest (PC 8/1)	24.17	0.27	2131 bp
fadK Digest (SK 8/1)	43	0.73	3717 bp
T4 Ligase Buffer	-	1	-
T4 Ligase	-	0.5	-
dH₂O	-	6.17	-

August 2, 2018

Planning Gibsons for Tomorrow

Sricharan

phz (2x insert excess)
Note for all the Gibsons we’re using new gBlocks that have revised short and end sequences designed to work well w/ pSB1C3 … using NEB calculator.

Fragments with identifying attributes and amounts listed below:

pSB1C3 (SK 7/30)	phz Start 7.27.2018	1F-3R (7/27)	phz3	4F-6R (8/1)	phz End 7.27.2018
50ng	gBlock, 10 ng/µL	24.6 ng/µL	gBlock, 10ng/µL	95.3 ng/µL	gBlock, 10ng/µL
20µL/375ng	426 bp	3072 bp	222 bp	2588 bp	277 bp
2.66µL	20.6ng	148 ng	10.7ng	125 ng	13.4 ng
0.037pmol	2.06µL	6.02µL	1.07µL	1.31 µL	1.34 µL

14.46µL total, add equivalent amt of NEB Gibson Master Mix

We’ll also do a ctrl w/ 50ng plasmid only w/ rest dH2O and NEB MMix
acr (2x insert excess)

Fragments with identifying attributes and amounts listed below:

pSB1C3 (SK 7/30)	acr1 7.27.2018	acr2	acr3 7.27.2018
25ng	gBlock, 10ng/µL	gBlock, 10ng/µL	gBlock, 10ng/µL
1.33µL	512 bp	529 bp	653 bp
	12.4ng	12.8ng	15.8ng
	1.24µL	1.28µL	1.58µL

5.43µL total, add equivalent amt of SGI Gibson MMix

pmt1231

Fragments with identifying attributes and amounts listed below:

pSB1C3 (SK 7/30)	pmt1231.1	pmt1231.2
25ng	gBlock, 10ng/µL	gBlock, 10ng/µL
1.33µL	532 bp	512 bp
	12.9ng	12.4ng
	1.29µL	1.24µL

3.84µL total, add 1.14µL dH₂O and 5µL SGI Gibson MMix

Also doing control with 25ng plamsid only and rest dH2O and SGI MMix
(1.33µL pSB1C3, 3.67µL dH₂O, 5µL SGI MMix)

August 2, 2018

Transformation (RbCl JM109) of 3A’s from 8/1

Peter

**See Protocol from 7/16/18**

Transforming:

CymA + J23110 + PSB1K3 (8/1 SK)
mtrCAB w/ new st. + J23110 + PSB1C3 (8/1 SK)
ccmA-H + B0015 + PSB1K3 (8/1 SK)
fadK + pTac + PSB1K3 (8/1 SK)
fadD ligation to PSB1C3 (7/24 LCC)

Notes/ Deviations:

Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube. Exceptions: 2 x 1μL for CymA and Mtr (from 20 μL 3A so more dilute than std 10 μL 3A); 2 x 1μL for fadD (hasn’t worked yet so adding a lot of DNA)
Heat-shocked for 30 seconds
Pooled like tubes (same vector) after 1 hour incubation
Plated with spinner and flame

Results (8/3/18):

Part/Vector	Transformation Result
CymA + J23110 + PSB1K3 (8/1 SK)	Good growth (O.C. on 8/3)
mtrCAB w/ new st. + J23110 + PSB1C3 (8/1 SK)	No growth
ccmA-H + B0015 + PSB1K3 (8/1 SK)	Good growth (O.C. on 8/3)
fadK + pTac + PSB1K3 (8/1 SK)	~32 colonies (O.C. on 8/3)
FadD (from ligation + now purified w/ PCR purif)	3 colonies (O.C. on 8/3)

August 3, 2018

Gibsons

Sricharan

Set up Gibsons as detailed 8/2/2018
Set @ 50°C 1 hr

4°C infinite hold

In thermal cycler (2 different cyclers for differing rxn volumes)

August

August 2, 2018

Transformation (RbCl JM109) of 3A’s from 8/1

Peter

**See Protocol from 7/16/18**

Transforming:

mtrCAB w/ new st. + J23110 + PSB1C3 (8/1 SK)
Phz Gibson (NEB) w/ redesigned start and end (8/3 SK)
Acr Gibson (SGI) w/ redesigned start and end (8/3 SK)
Pmt1231 Gibson (SGI) w/ redesigned start and end (8/3 SK)
NEB PSB1C3 ctrl
SGI PSB1C3 ctrl

Notes/ Deviations:

Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube. Exceptions: 2 x 1μL for Mtr (from 20 μL 3A so more dilute than std 10 μL 3A)
Heat-shocked for 30 seconds
Pooled like tubes (same vector) after 1 hour incubation
CAM plates for all

Results (8/7/18):

Part/Vector	Transformation Result
mtrCAB w/ new st. + J23110 + PSB1C3 (8/1 SK)	No growth
Phz Gibson (NEB) w/ redesigned start and end (8/3 SK)	No growth
Acr Gibson (NEB) w/ redesigned start and end (8/3 SK)	No growth
Pmt1231 Gibson (SGI) w/ redesigned start and end (8/3 SK)	5-10 colonies (O.C. on 8/8)
NEB PSB1C3 ctrl	No growth
SGI PSB1C3 ctrl	No growth

August 7, 2018

Minipreps of 3As and fadD + Gel

Sricharan

Using Genscript Kit miniprepped:

tac+fadK 3A
cymA 3A (w/ med Anderson)
B0015+ccmA-H 3A
fadD

Nanodropped, results below:

Sample Name	Concentration (ng/μL)	260/280	260/230
tac+fadK	4.08	1.51	1.16
cymA 3A	41.13	1.58	0.69
B0015+ccmA-H	59.52	1.61	0.77
fadD	156.54	1.82	1.90

Diagnostic digests of these items, with

dH₂O	0μL
CutSmart	2μL
DNA	17μL
EcoRI	1μL

August 7, 2018

Gel of a bunch of things

Liz

Made a 1% agarose gel

1	2	3	4	5
Ladder (6μL)	Tac + FadK EcoRI digest (5μL sample + 1μL digest)	cymA 3A EcorI digest (5μL sample + 1μL digest)	ccmA-H + B0015 3A EcoRI digest ((5μL sample + 1μL digest)	fadD EcoRI digest (5μL sample + 1μL digest)

Only lane 5 (fadD) had a band

August 8, 2018

Digests for 3A Assemblies

Liz, Peter

Double digests of pSB1A3 and J23114

pSB1A3:

cutsmart	2μL
DNA	15μL
EcoRI	1μL
PstI	1μL
DpnI	1μL

DNA from iGEM linearized plasmid backbone

In retrospect this is bad because need water in the digestion

J23114:

cutsmart	2μL
DNA (J23114 miniprep SD 7/25)	10μL
EcoRI	1μL
SpeI	1μL
water	6μL

Heat blocked at 37C for 15 min
Heat blocked at 80C for 15 min
Assembly part is in Peter’s notebook

August 8, 2018

3A Assemblies

Peter

***Using NEB Ligation Calculator for amounts, 1:1:1 molar ratios***

Protocol:

Add components in following order: water, buffer, DNA, enzymes
Let sit at room temp for 10 minutes
Heat block @ 65 C for 20 minutes

mtrCAB w/ new st. + Anderson (J23114) + pSB1C3

Component	Mass (ng)	Volume (µL)	Notes
pSB1C3	25	0.68	(7/30 SK)
J23114 Digest	36.03	0.21 (should have been 0.33)	(8/8 LCC)
mtrCAB w/ new st.	89.58	2.86	(8/1 SK)
T4 Ligase Buffer	-	2	-
T4 Ligase	-	1	-
dH₂O	-	3.26	-

mtrCAB w/ new st. + pTac (K864400) + pSB1A3

Component	Mass (ng)	Volume (µL)	Notes
pSB1A3	25	1.25	(8/8 LCC)
pTac (K864400)	24.72	0.27	(SK 8/1)
mtrCAB w/ new st	2.74	2.86	(SK 8/1)
T4 Ligase Buffer	-	2	-
T4 Ligase	-	1	-
dH₂O	-	2.74	-

August 8, 2018

Transformations of Gibsons and 3A Assemblies

Liz and Sricharan

1	2	3	4	5	6	7
NEB control gibson pSB1C3 (SK 8/3)	SGI control gibson pSB1C3 (SK 8/3)	Acr Gibson (SK 8/3)	Phz Gibson (SK 8/3)	mtrCAB with new start and J23114 pSB1C3 (PC 8/8)	mtrCAB with new start and pTac (K864400) in pSB1A3 (PC 8/8)	mtrCAB with new start and J23110 in pSB1C3 (SK 8/1)

2 tubes per sample, 1μL DNA in each tube
Followed transformation protocol (iGEM)

August 8, 2018

Colony PCR Checks of 3A Assemblies

Liz and Sricharan

1st Thing: Setting up PCR mixes … Fprimer = VF2, Rprimer = VR @ 10μM

Taq PCRs

Item	GoTaq (stuff < 3kb) … 100μL rxn
5x Buffer	20μL
10mM dNTPs	2μL
F Primer	5μL
R Primer	5μL
dH₂O	62.5μL
GoTaq Polymerase	0.5μL
Colony Suspensionr	5μL

	Temperature (°C)	Time
Initial Denaturation	95	5min
35 Cycles	95	45sec
	65	45sec
	72	2.5min
Final Extension	72	5min
Soak	4	Infinite

Phusion PCRs

Item	NEB Phusion (ccm) … 50μL
5x Buffer	10μL
10mM dNTPs	1μL
F Primer	2.5μL
R Primer	2.5μL
dH₂O	31μL
GoTaq Polymerase	0.5μL
Colony Suspension	2.5μL

	Temperature (°C)	Time
Initial Denaturation	98	5min
35 Cycles	98	10sec
	63	30sec
	72	2:10min
Final Extension	72	10min
Soak	4	Infinite

Picked 4 colonies of each 3A assembly (B0015+ccmA-H, tac+fadK, med Anderson+cymA)

Put tip in 20μL dH2O, pipetted up and down, spotted 3μL onto marked plate (Cam), incubated overnight … Later realized that these plasmids are kanamycin resistant

Put 0.5μL w/9.5μL of master mix above … ran cycler

Will run gel to check which ones are right

August 8, 2018

Colony PCR Checks of 3A Assemblies

Sricharan and Liz and Shannon

Lane	1	2	3	4	5
Item	2-log ladder	ccm colony 1	ccm colony 2	ccm colony 3	Phusion colony ctrl

Run @ 100V, 25min
No visible bands on this gel (?)

Lane	1	2	3	4	5
Item	2-log ladder	fadK colony 1	fadK colony 2	fadK colony 3	fadK colony 4

Lane	6	7	8	9	10
Item	cymA colony 1	cymA colony 2	cymA colony 3	cymA colony 4	GoTaq colony control

Run @ 100V, 25min

August 9, 2018

Transformations & OCs

Sricharan and Peter

Usual PC protocol

acr Gibson 8/3 (Strataclone, 1 tube, 1)
phz Gibson 8/3 (Strataclone, 1 tube 1)
weak Anderson + mtrCAB revised 8/8
acr Gibson 8/3 (RbCl cells)
phz Gibson 8/3 (RbCl cells)
pTac + mtrCAB revised 8/8

3, 4, 5, 6 - 2 tubes RbCl cells (50μL aliquots), 1μL DNA each tube

OCs of following in Cam:

tac+fadK 3A colony PCR (8/8)
pmt1231 Gibson (8/6)
med Anderson + mtrCAB w/ new start (8/8)

August 13, 2018

Transformation of anderson, araC, and pBad/ araC promoters

Peter

**See Protocol from 7/16/18**

Transforming:

BBa_J23112 (amp)
BBa_J23103 (amp)
BBa_J23109 (amp)
BBa_J23117 (amp)
BBa_R0080 (inducible araC)
BBa_I0500 (inducible pBad/ araC)

Notes/Deviations:

Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube
Heat-shocked for 30 seconds
Pooled like tubes (same vector) after 1 hour incubation

August 13, 2018

OCs

Sricharan

OCs of pmt1231, acr, phz Gibsons - 2 colonies each
cymA, tac+fadK 3As - 3 colonies each
All OCs grew after 18 hours

August 14, 2018

Making Ampicillin Plates + Diagnostic Digests

Sricharan

From LCC’s MPs - Nanodropped - 3As are low concentration (~10-20ng/μL, Gibsons high concentration ~100ng/μL)

For digests:

Item	3A Assemblies	Gibsons
dH₂O	0μL	12μL
CutSmart	2μL	2μL
DNA	17μL	5μL
EcoRI-HF	1μL	1μL

August 14, 2018

Lots of minipreps

Liz

Did 12 minipreps to digest and run on a gel

Pmt1231 (1 and 2)
Acr gibson (1 and 2)
Phz gibson
Tac and fadK 2A (1, 2, 3)
cymA 3A

Used GenScript kit and eluted in 30μL elution buffer

Minipreps were digested and run on a gel

Only pmt1231 had a logical band

Made a glycerol stock with 0.75mL 40% glycerol and 0.75mL pmt overnight culture

August 15, 2018

more minipreps

Liz

Did minipreps of overnight cultures

BBa_R0080
BBa_J23103

Used GenScript kit

Eluted in 30μL elution buffer

Nanodropped:

BBa_R0080: 113.97 ng/μL
BBa_J23103: 307.66 ng/μL

August 15, 2018

Colony PCR Gel

Sricharan and Liz

More colony PCRs using same protocol as some weeks before, this time using the correct antibiotic selection plate.

August 16, 2018

Colony PCR Gel

Sricharan and Liz

All samples had 5μL DNA and 1μL loading dye

Gel 1:

1	2	3	4	5	6	7	8	9	10	11
ladder	Mtr 1	Mtr 2	Mtr 3	Mtr 4	Mtr 5	Mtr 6	phz1	phz2	phz3	ladder
12	13	14	15	16	17	18	19	20	21	22
phz4	phz5	phz6	ccm1	ccm2	ccm3	ccm4	ccm5	ccm6	Kan phusion ctrl	ladder

Gel 2:

1	2	3	4	5	6	7	8	9	10	11
ladder	fadK1	fadK2	fadK3	fadK4	fadK5	fadK6	acr1	acr2	Kan ctrl gotaq	ladder
12	13	14	15	16	17	18	19	20	21	22
Cam ctrl gotaq	empty	cymA1	cymA2	cymA3	cymA4	cymA5	cymA6	Cam ctrl phusion	ladder	empty

August 16, 2018

phz Linear Gibson PCR

Liz

Thermocycler

	time	Temperature (C)
Initial denaturation	30 s	98
35 cycles	10 s	98
	20 s	64
	2:10 (min:sec)	72
Final extension	10 min	72
hold	infinite	4

PCR tube

Phusion HF buffer	4μL
DMSO	0.6μL
10mM 1F primer	1μL
10mM 6F primer	1μL
dNTPs	0.4μL
Template DNA (used SK linear gibson)	1μL
phusion	0.2μL

August 24, 2018

Restriction Digests for 3A Assemblies and 3A Assembly

Liz

cymA: cymA + J23110 (medium Anderson promoter) + pSB1K3

J23110

water	13.79μL
CutSmart	2μL
DNA (J23110 miniprep 7/27 LCC)	300ng = 2.21μL
EcoRI	1μL
SpeI	1μL

cymA

water	13.51μL
CutSmart	2μL
DNA (cymA miniprep SD 7/25)	300ng = 2.49μL
EcoRI	1μL
PstI	1μL

pSB1K3

water	4μL
CutSmart	2μL
DNA (J23110 miniprep 7/27 LCC)	300ng = 12μL
EcoRI	1μL
PstI	1μL

Put in heat block at 37C for 15 min
Heat killed at 80C for 15 min

Forgot that backbone also needs to be digested with DpnI so cooled, added 1μL DpnI to pSB1K3, heatblocked at 37C for 15 min, and heat killed at 80C for 15 min

Used digestions in 3A assembly:
cymA + J23110 + pSB1K3 (used open wetware protocol)

water	2.17μL
Digested pSB1K3	10ng = 0.7μL
Digested cymA	37.02ng = 2.45μL
Digested J23110	47.75ng = 3.18μL
T4 ligase buffer	1μL
T4 ligase	0.5μL

Thermocycler:

22.5C for 30 min
65C for 10 min

August 24, 2018

A lot of colony PCR fails and Linear Gibson

Sricharan

A new Linear Gibson of phz 1F-6R

5μL	NEB MMix
3.01μL	1F-3R (7/27)
0.53μL	phz3 (gBlock)
0.66μL	4F-6R (8/1)
0.80μL	dH₂O

Running PCR on this Gibson (Phusion) using usual protocol, and using 1μL of template. Annealing temperature is 64 degrees C, extension time is 2min,30sec.

August 27, 2018

cymA 3A gel

Sricharan and Peter

Changing some items to potentially have success in Gibson reactions:

Also redesigned inserts so that there are correct overlaps with pSB1C3

Using 0.05pmol backbone, equimolar inserts

pSB1C3 (SK 7/30)	acr1 7.27.2018	acr2	acr3 7.27.2018
3.57μL	gBlock (532bp)	gBlock (529bp)	gBlock (653bp)
67ng	16.57ng	17.12ng	21.14ng
2070bp	1.66μL	1.71μL	2.11μL

pSB1C3 (SK 7/30)	phz Start 7.27.2018	phz 1F-6R	phz End 7.27.2018
3.57μL	gBlock (426bp)	gBlock (5882bp)	gBlock (277bp)
67ng	13.79ng	190.4ng	8.97ng
2070bp	1.38μL	2.73μL	0.90μL

For actual reactions:

Cutting reaction volume in half (5μL Master mix, 5μL DNA + water)

August 27, 2018

phz 1F-6R Gel

Liz

Made a 1% agarose gel for linear phz 1F-6R gibson PCR

Purified linear 1F-6R gibson PCR (SK 8/24) using lambda kit

Nanodropped: 69.7 ng/μL

Gel:

1	2	3	4
ladder	Phz gibson pure PCR	pSB1C3 A	pSB1C3 B
	5μL sample + 1μL dye	1μL dye+ 1μL sample + 4μL water

August 28, 2018

Linear Gibson PCR + Making more Backbone

Sricharan

Running Phusion PCR on phz 1F-6R (before the PCR was ~1kb, should have been ~6kb), pSB1C3

Usual protocol: for phz used 0.4μL sample in 20μL rxn, annealing at 64 degrees C, extension 2 minutes. Did PCR in HF and GC Buffers
For pSB1C3, used 0.2μL of Peter’s diluted J04450 MP (7/24) to 0.14ng/μL (100x). This was a 50μL rxn, annealing at 71 degrees C, 40s extension.

PCR purified on 8/28 by Sricharan

Nanodrop

	phz HF	phz GC	pSB1C3 HF
Conc (ng/μL)	15.1	21.7	39.5
260/280	1.43	1.63	1.71
260/230	-0.27	-0.48	-0.49

Curves look like junk for all 3
Peter has gel on pg 49

August 28, 2018

Gel of Phz (1F-6R) pcr’s and pSB1C3

Peter

Lane	1	2	3	4
Item	2-log ladder	- 3 μL Phz (HF) pcr -2 μL dH₂O -1 μL loading dye	Phz (GC) pcr -2 μL dH₂O -1 μL loading dye	pSB1C3 (HF) -2 μL dH₂O -1 μL loading dye

August 28, 2018

Transformation (RbCl JM109) of 3A’s from 8/1

Peter

**See Protocol from 7/16/18**

Transforming:

Acr circular gibson (8/27 SK)
CymA + J23110 + PSB1K3 3A (8/24 LCC)

Notes/Deviations

Two tubes of cells/ transformation (to ensure one tube will have cells) so 0.5 μL of DNA added to each tube. Exceptions: 2 x 1μL for Mtr (from 20 μL 3A so more dilute than std 10 μL 3A)
Heat-shocked for 30 seconds
Pooled like tubes (same vector) after 1 hour incubation
CAM for Acr
Kan for CymA

August 30, 2018

Overnight Cultures of cymA 3A Assemblies

Liz

Overnights of cymA 3A assemblies
Took 3 colonies from “RbCl transformation cymA + J23110 + pSB1K3 3A PC 8/28” plate

3A done by LCC 8/24

5mL LB broth + 5μL kanamycin
Put in 37C shaker overnight

August 31, 2018

Miniprep of Cultures Liz Picked up this Morning

Sricharan

MP of cymA + J23110 + pSB1K3 by LCC … Genscript Kit, eluted 30μL and glycerol stocks

September

September 4, 2018

cymA 3A gel

Liz

Made 1% agarose gel

Nanodropped SK’s cymA minipreps 1-3 (8/31)

Miniprep 1: 21.05 ng/μL
Miniprep 2: 17.49 ng/μL
Miniprep 3: 29.24 ng/μL

Gel:

1	2	3	4
Ladder (6μL)	cymA miniprep 1	cymA miniprep 2	cymA miniprep 3

Samples loaded with 5μL sample and 1μL loading dye

Didn’t digest before running gel--ran again next day

September 5, 2018

cymA 3A gel 2.0

Liz

Digested cymA minipreps with EcoRI to run on gel

CutSmart	2μL
DNA (cymA minipreps 1-3 SK 8/31)	12μL
EcoRI	1μL
water	5μL

Heat blocked at 37C for 15 min

Heat killed at 65C for 15 min

Ran 1% agarose gel (same setup as previous day)

September 5, 2018

Gel extraction of phz

Peter

Notes:

Following Azady’s advice on getting greater concentration of pieces for gibson

Gel:

1	2
Ladder (6μL)	10 μL phz 4F-6R pure pcr (PC 7/13)

Extraction:

Bio basic kit
400 μL buffer * (0.048 g/100 mg) * (1000 mg/1 g) = 192 μL buffer used

September 7, 2018

phz 4F-6R PCRs - Aiming for high purity

Sricharan

Using

Original Gibson (7/12)
Old gel extract
PC new gel extract (9/5)

Usual Phusion PCR, 1μL template DNA, 67 degrees C annealing, 1:18 extension time. All put into 1.5mL tubes

September 10, 2018

PCR purification, Nanodrop, Gel

Ryan

Started off by preparing a small gel, .125g agarose, 12.5 mL TAE, mixed and microwaved for 30 seconds. Added 1.25 uL SYBR Safe and poured into mold and let sit

PCR purification of Charan’s 3 PCR products from 9/3/18, phz 4F-6R

Volume of PCR product was 20 uL, so added 100 ul Binding Buffer, and loaded it into Spin column and centrifuged for 1 min, @10,000 rpm
Discard flow-through and add 700 uL Wash Buffer, centrifuge again (same settings)
Discard flow-through, centrifuge again
Transfer top of spin column into a new 1.5 mL tube for collection
Add 40 uL Elution Buffer and let sit for 1 min
Centrifuge again

I labelled the 3 PCR products 1, 2, 3 and ran a nanodrop

1 was found to be 22.8 ng/uL
2 was found to be 19.1 ng/uL
3 was found to be 16.5 ng/uL

Put gel into the electrophoresis machine and added 6 uL 2-log Ladder into lane 1

3 uL of PCR 1 + 2uL water + 1 uL loading dye into lane 2
3 uL of PCR 2 + 2uL water + 1 uL loading dye into lane 3
3 uL of PCR 3 + 2uL water + 1 uL loading dye into lane 4

And ran it for 30 minutes at 100V *23 minutes dye was near end of gel

September 10, 2018

Azady PCRs acr3 and pXB1C3

Liz

Acr

HF buffer	4uL
10mM dNTPs	0.4uL
F primer (Azady acr 3F)	1uL
R primer (Azady acr 3R)	1uL
Template DNA (acr 3 gBlock)	0.2uL
DMSO	0.6uL
phusion	0.2uL
Water	12.6uL

pSB1C3

HF buffer	4uL
10mM dNTPs	0.4uL
F primer (SB-prep-3P-1-mutated)	1uL
R primer (SB-prep-2Ea)	1uL
Template DNA (BBa_R0080 diluted 10x)	0.2uL
DMSO	0.6uL
phusion	0.2uL
Water	12.6uL

Thermocycler

Initial denaturation	98C	30s
35 cycles	98C	10s
	67C	30s
	72C	35s
Final extension	72C	10 min
hold	4C	infinite

September 13, 2018

Azady PCRs, acr 1 & 2

Sricharan

PCRing parts of acr to remove hairpins and amplify gBlocks, for better Gibson results

Resuspended primers in TE

spun down dry tubes
to 100µM in tubes, let sit 10 min
aliquot and dilute to 10µM

Usual Phusion HF Buffer
For acr 1, Primers: Azady acr 1F + Azady acr 1R, Anneal 68°C
For acr 2, Primers: Azady acr 2F + Azady acr 2R, Anneal 67°C
Usual cycle, with 15s extension time for both

Transferred into 1.5mL tubes

September 13, 2018

PCR Purifs + Gel of acr 1-3 + pSB1C3 mut

Sricharan

PCR purifying acr 1-3 gBlock PCRs & pSB1C3 mut (adapted for Gibson)
Lambda Kit, eluting with 30µL … Nanodrop and gel by PC

September 14, 2018

Gel of Acr g-block PSB1C3 pcr’s

Peter

Notes:

Following Azady’s advice to pcr the g-blocks and backbone before gibson
Backbone is mutated (using primers with one nucleotide difference) so can overlap with new acr 1 & 3 g-blocks and not form hairpin

Gel:

1	2	3	4	5
Ladder (6μL)	Acr 1 pcr purif. (9/14 SK) -512 bp	Acr 2 pcr purif. (9/14 SK) -529 bp	Acr 3 pcr purif. (9/14 SK) -653 bp	Mutated pSB1C3 pcr purif. (SK 9/14)

September 17, 2018

Azady PCR acr 1 and 2 (more DNA)

Liz

HF buffer	4uL
10mM dNTPs	0.4uL
F primer (Azady acr 1F/2F)	1uL
R primer (Azady acr 1R/2R)	1uL
Template DNA (acr 1, 2 gBlock)	2uL
DMSO	0.6uL
phusion	0.2uL
Water	10.8uL

Temperatures for thermocycler from Sricharan’s same PCR last week

September 18, 2018

Zymo DNA concentrator planning (acr)

Peter

Notes:

Concentrating fragments before gibson as suggested by Azady
Wanted 50 ng of acr 3 and calculated equimolar amounts of other fragments (acr 1 & 2)
Used 2:1 fragment to backbone ratio
Need to add at least 6 μL elution buffer
Added total of 10 μL of elution buffer to get 8.7 μL final product
Below is initial amounts added to column, see zymo kit for further details on the next steps and buffers to add
Centrifuging done at 15500 g

Component	Amount (μL)
Acr 3 pcr purif. (9/14 SK)	5.62
Acr 1 pcr purif. (9/14 SK)	3.25
Acr 2 pcr purif. (9/14 SK)	2.57
Mutated pSB1C3 pcr purif. (9/14 SK)	7.86
Binding Buffer	96.5 = 19.3 * 5 (total volume DNA mult. 5)

September 18, 2018

Gibson of acr (circular)

Peter

Component	Amount (μL)
Zymo pcr fragments product	8.7
SGI GA HiFi Master Mix	8.7

-run in thermo cycler @ 50 C for 1 hour

September 19, 2018

Transformation of acr Gibson (by PC)

Sricharan

We’ve now PCRed parts for acr Gibson and backbone (some mutations to remove secondary structure)

Peter concentrated DNA and ran Gibson yesterday
Note: (By accident) we forgot to restriction digest pSB1C3 mut
Will this affect product?
Also no - control with backbone … will do - control with water

Transformation of the usual RbCl2 cells with 1μL in each 50μL cells tube

Acr - transformed with 1μL acr Gibson each 50μL cells
Ctrl - transformed with 1μL dH2O each 50μL cells … 1 tube was forgotten so only transformed tube

Plates labeled PC since Peter helped

September 19, 2018

Azady PCR: acr 3 and pSB1C3 (2nd time)

Sricharan

Acr 3: Phusion HF PCR with 67°C annealing, 15 sec extension, primers Azady acr 3F and azady acr 3R
pSB1C3: Phusion HF PCR with 66°C annealing, 40 sec extension, primers SB-prep-3P-1-mut and SB-prep-2-Ea

September 24, 2018

Acr 1-3 PCR cleanup and gel

Liz

Made 1% agarose gel (small wells)

PCR purified acr 1-3 and pSB1C3

Acr 1-2 LCC 9/17
Acr 3 and pSB1C3 mut SK 9/21
Lambda kit
Eluted in 30μL

Nanodropped:

Acr 1: 12.68 ng/μL
Acr 2: 19.82 ng/μL
Acr 3 HF: 33.59 ng/μL
Acr 3 GC: 9.76 ng/μL
pSB1C3: 78.88 ng/μL

Gel:

1	2	3	4	5	6	7	8
ladder	Acr 1	Acr 2	Acr 3 HF	Acr 3 GC	pSB1C3	ladder	Acr 1
6μL	5μL sample, 1μL dye					6μL	1μL dye, 1.5μL water, 3.5μL sample

September 26, 2018

Transforming acr

Sricharan

Trying again to transform Gibson acr from 9/17

1 tube (50μL) Strataclone - 4μL acr Gibson (9/17)
2 tubes (100μL) RbCl cells - 8μL acr Gibson (9/17)

1 tube (50μL) RbCl cells - 4μL dH₂O (ctrl)

Usual protocol for RbCl cells
For Strataclone
20 min on ice
45 sec heat shock
2 min on ice
250μL SOC - Incubate slightly >1hr
Plate 100μL or so

Tubes started 1 hr incubation at 2:45pm
End 3:45 pm

3 plates waiting on bench for Peter to plate - not in warm room since it seems humid there

September 27, 2018

acr OCs

Sricharan

Picked up transformations from yesterday
1 colony RbCl acr
Many (~20 colonies) Strataclone acr

OC’ed 1 RbCl and 6 Stratclone colonies

LB from Kate downstairs (says it was made 2013 on bottle?)
Accidentally forgot to add Cam so added after tips were submerged in tubes
In at 4:38pm, shaker downstairs

September 28, 2018

acr Miniprep/Diagnostic

Liz

Made 1% agarose gel

Minipreps of strataclone acr 1-6 and RbCl acr (SK 9/27)

Used genscript kit
Eluted in 30μL elution buffer

Single digest for diagnostic gel

CutSmart	2μL
water	7μL
Miniprep DNA (str acr 1-6 and RbCl acr)	10μL
EcoRI	1μL

Thermocycler:

37C for 15 minutes
65C for 15 min
4C hold

Gel:

1	2	3	4	5	6	7	8
ladder	Acr str 1	Acr str 2	Acr str 3	Acr str 4	Acr str 5	Acr str 6	RbCl acr

5μL sample, 1μL loading dye

	1	2	3	4	5	6	7	8	9	10	11	12
A	497	266	144	75	41	23	14	9	8	6	6	5
B	532	302	153	81	44	24	25	10	7	7	5	5
C	532	295	154	81	45	25	15	10	7	7	6	5
D	547	297	159	83	45	25	16	11	8	7	6	5

	1	2	3	4	5	6	7	8	9	10	11	12
A	497	266	144	75	41	23	14	9	8	6	6	5
B	532	302	153	81	44	24	25	10	7	7	5	5
C	532	295	154	81	45	25	15	10	7	7	6	5
D	547	297	159	83	45	25	16	11	8	7	6	5

Team:UConn/Notebook

Notebook

Interlab

OD

CFU

Dilution 3

Dilution 4

Dilution 5

Lab Notebook

May

May 22, 2018

Competent Cell Prep

Liz, Peter, Ryan, Shannon, Sricharan

TfbII (50 mL)

TfbI (200 mL)

ᴪa (500 mL)

May 22, 2018

Interlab Calibration

Liz, Peter, Ryan, Shannon, Sricharan

Calibration 1, OD600 Reference Point - LUDOX protocol:

Absorbances

Calibration 2

Absorbance

Calibration 3: Fluorescence Standard Curve

Fluorescein stock solution:

Fluorescence

May 23, 2018

Competent Cell Prep

Sricharan

May 24, 2018

Competent Cell Prep

Liz, Peter, Shannon, Sricharan

May 24, 2018

Competent Cell Prep

Liz, Peter, Shannon, Sricharan

May 29, 2018

Interlab Transformation

Shannon, Liz

Plasmid Re-suspension

May 29, 2018

Chloramphenicol Plate Preparation

Ryan, Peter, Sricharan

Preparing Chloramphenicol Stock

Preparing Chloramphenicol LB Plates (24)

May 30, 2018

Interlab Transformation

Shannon, Liz, Ryan

May 30, 2018

Competent Cell Transformation (Scott-Simanis, Test Kit)

Peter, Sricharan

May 31, 2018

LB + Chloramphenicol Media Preparation (w/o agar)

Peter, Sricharan, Liz, Shannon, Ryan

Preparing Chloramphenicol LB media (500 mL)

May 31, 2018

Interlab Overnight Cultures

Peter, Sricharan, Liz, Shannon, Ryan

June 1, 2018

Interlab Fluorescence Measurements

Peter, Sricharan, Liz, Shannon, Ryan, Toni

June 1, 2018

LB + Chloramphenicol Plates Preparation

Peter, Ryan

Preparing Chloramphenicol LB plates (24)

June 1, 2018

Title

Names

June 1, 2018

Title

Sricharan, Liz

June

June 4, 2018

LB + Chloramphenicol Plate Prep

Ryan

Notes:

June 4, 2018

Interlab Transformation of 21B and 21D

Liz

Deviations/Notes

Times

Calibration 1, OD₆₀₀ Reference Point - LUDOX protocol:

OD₆₀₀

Culture Volume (checked OD₆₀₀ with plate reader)

New Protocol - we were doing stuff wrong

See protocol from 6/1/18

See protocol from 6/12 (week 5)

	1	2	3	4	5	6	7	8	9	10	11	12
A	497	266	144	75	41	23	14	9	8	6	6	5
B	532	302	153	81	44	24	25	10	7	7	5	5
C	532	295	154	81	45	25	15	10	7	7	6	5
D	547	297	159	83	45	25	16	11	8	7	6	5