Day 1, Monday 11/06

People In Lab:Amalia, Carla, Daniel, Jasmeen, Nina, Tashi

Agenda:

• Transform 2J interlab plasmid into DH5α and plate on LB+CAM

• LB+CAM plates

• Interlab calibration #2: Particle Standard Curve - Microsphere Protocol

• Electroporate MG1655ΔlacI

• Transform pSB1C3+RFP and pET28a+arg1 into MG1655ΔlacI

• Make and autoclave PBS for interlab calibration #3

• Autoclave double distilled water

Equipment used:

• Autoclave

• Bunsen burner

• Plate reader

• Thermomixer

• 37°C incubator in room 301

• Electroporator

Protocol: iGEM protocols used for transformation, electroporation, LB+agar. Interlab protocol used for Interlab calibration #2: Particle Standard Curve - Microsphere Protocol.

Transformation of 2J plasmid

1μLof 2J plasmid used, 50μLof cells plated

Interlab calibration #2

• 200μLbead stock in wells A1, B1, C1, D1

• 100μLdouble distilled water in wells A2-A12, B2-B12, C2-C12, D2-D12

• performed serial dilution by transferring 100μLeach time, until well A11, B11, C11, D11

• used the plate reader to shake the plate at an amplitude of 3.5 for 7 seconds, and measure the absorbance at wavelength 600nm

Double transformation of pSB1C3+RFP and pET28a+arg1 into MG1655ΔlacI

• 2μLof pSB1C3+RFP(10pg/μL)

• 1μLofpET28a+arg1

• plated 100μLon LB+CAM+KAN

• plated 200μLon LB+CAM+KAN

• plated 300μLon LB+CAM+KAN

• plated negative control (100μLof untransformed MG1655ΔlacI)

Next Steps:

• Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol

• Interlab cell measurement protocol

Day 2, Tuesday 12/06

People In Lab: Amalia, Carla, Daniel, Nina

Agenda:

• Second attempt at pSB1C3+RFP and pET28a+arg1 double transformation in MG1655ΔlacI, testing for incompatibility

• Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol

• Make overnight cultures from interlab transformation plates

Equipment used:

• Bunsen burner

• Plate reader

• Electroporator

• Shaker in room 315

• 37°C incubator in room 301

Protocol: iGEM protocols used for transformation by electroporation. Interlab protocol used for for Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol

Double transformation of pSB1C3+RFP and pET28a+arg1 into MG1655ΔlacI (electroporation)

Hypothesis: If we get growth on the positive control, LB+KAN, and LB+CAM plates with transformed cells, but not the double antibiotic plate, we can say that either the plasmids are incompatible, or the double antibiotic plate concentration is too high.

• 1.5μLof RFP (50pg/μL)

• 1.5μLof pET28a+arg1

• plated 150μLof transformed cells on LB+CAM+KAN

• plated 100μLof transformed cells on LB+CAM

• plated 100μLof transformed cells on LB+KAN

• plated 10μLof untransformed cells on LB+KAN (negative control)

• plated 10μLof untransformed cells on LB (positive control)

Interlab calibration #3

• serial dilution was performed and the plate was read

• shaken 5s at amplitude = 2

• excitation = 485nm

• emission = 525nm

• optimal gain used = 75

• read from bottom of plate

• The graph obtained from this experiment indicated an error, will repeat tomorrow

Interlab transformation overnight cultures

• 16 tubes with 5mL of LB and 5μLCAM

• 2 colonies were taken from each of the 8 plates (2B, 2D, 2F, 2H, 2J, 2L, 2N, 2P)

• tubes placed in the shaker overnight at 37° at 250rpm

Observations:

Double transformation of pSB1C3+RFP and pET28a+arg1 into MG1655ΔlacI

• no colonies on negative control plate

• no colonies on 100μL, 200μL, or 300μLplates

100μL double transformation of pSB1C3+RFP (10pg/μL) and pET28a+arg1 in MG1655ΔlacI (negative control) on LB+CAM+KAN

100μL double transformation of pSB1C3+RFP (10pg/μL) and pET28a+arg1 in MG1655ΔlacI on LB+CAM+KAN

200μL double transformation of pSB1C3+RFP (10pg/μL) and pET28a+arg1 in MG1655ΔlacI on LB+CAM+KAN

300μL double transformation of pSB1C3+RFP (10pg/μL) and pET28a+arg1 in MG1655ΔlacI on LB+CAM+KAN

Potential reasons for results:

• antibiotic concentration too high (50μL/mL KAN + 25μL/mL CAM)

• MG1655ΔlacIcells may no longer be competent

• left MG1655ΔlacIcells thawing in the ice for too long

• electroporation might have failed

• plasmids may not have been compatible

• we could try sequential transformation (still have to consider antibiotic concentration of LB+KAN+CAM plate)

Next Steps:

Finish Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol

Cell growth, sampling, assay (interlab cell measurement)

Day 3, Wednesday 13/06

People In Lab:Amalia, Carla, Daniel, Nina

Agenda:

• Redo Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol

• Autoclave glass bottles, beads, tips, eppendorf tubes

• Reculture the overnight cultures from the interlab transformation

Equipment used:

• Autoclave

• Bunsen burner

• Plate reader

• 37°C shaker in room 315

Protocol: Used interlab protocol for interlab calibration.

Second attempt at Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol

• serial dilution was performed and the plate was read

• excitation = 485nm

• emission = 525nm

• optimal gain used = 90

• read from bottom of plate

Reculturing the overnight cultures from the interlab transformation

• 16 tubes with 5mL of LB and 5μLCAM

• 100μLfrom each previous overnight culture placed in respective new tube (DH5α)

• tubes slightly unscrewed and tape placed on lid for aeration in shaker

• tubes placed in the shaker overnight at 37°C at 250rpm

Observations:

Second double transformation pSB1C3+RFP and pET28a+arg1in MG1655ΔlacI

• LB+CAM+pSB1C3+RFP and pET28a+arg1

  • sparsely populated pink colonies

  • presence of some colonies

• LB+KAN+pSB1C3+RFP and pET28a+arg1

  • no visible colonies

• LB+KAN+CAM+pSB1C3+RFP and pET28a+arg1

  • no visible colonies

• LB+KAN+nontrasformed cells (negative control)

  • no visible colonies

• LB+nontransformed cells (positive control)

  • growth on entire plate

Second double transformation pSB1C3+RFP and pET28a+arg1 in MG1655ΔlacI

Potential reasons for these results:

• colonies on LB+CAM+pSB1C3+RFP+pET28a+arg1) because the cells are still competent and were transformed with pSB1C3+RPF

• no colonies on LB+KAN+pSB1C3+RFP and pET28a+arg1not because the cells are not competent, but rather the transformation (pET28a+arg1into MG1655ΔlacI) failed

• no colonies on LB+KAN+CAM+pSB1C3+RFP+pET28a+arg1 because the backbones are not compatible

• no growth on LB+KAN+non transformed cells (negative control) because there was no antibiotic resistance gene in the cells

• we had growth on LB+non transformed cells (positive control) because the cells were viable and there was no selection in the plate against them

  • pET28a = KAN resistant - incompatible with pSB1C3 by group check

  • pSB1C3 = CAM resistant - higher copy number than pET28a

Next Steps:

Finish interlab cell measurement protocol

Day 4, Thursday 14/06

People In Lab:Ahmed, Amalia, Daniel, Monica, Nina, Tashi

Agenda:

• Interlab cell measurement protocol

• Autoclave P1000 tips

• LB media

• LB+CAM plates

Equipment used:

• Autoclave

• Plate reader

• Shaker in autoclave room

• Bunsen burner

• 37°C incubator in room 301

Protocol: iGEM protocols used for LB, LB+agar. Interlab protocols used for interlab OD and CFU measurements.

Observations:

Next Steps:

• Count colonies on CFU cell measurement plates for interlab cell measurement protocol

• Transform pET28a+arg1intoMG1655ΔlacI cells

Day 5, Friday 15/06

People In Lab:Ahmed, Amalia, Nina, Tashi

Agenda:

• Check overnight plated cultures for interlab CFU count

• Transformation of pET28a+arg1 into electrically competent MG1655ΔlacIcells

Observations:

Interlab CFU count

Miniscule colonies on some plates after ~11 hours of incubation at 37°C. Some plates did not have any colonies. Plates were put back in the incubator for a few more hours.

Equipment used:

• 37°C incubator in room 301

• Shaker in room 315

• Electroporator

Protocol: iGEM protocols used for transformation by electroporation.

Transformation of ARG1 in electrically competent MG1655ΔlacIcells

• pET28a+arg1diluted from 39.3ng/mL to 1ng/mL

  • 38.3μLnuclease free water + 1μLpET28a+arg1

• transformed cells in cuvette placed in shaker at 37°C, 250rpm

• 3 plates

  • 40μLof untransformed MG1655ΔlacIcells (negative control)

  • 100μLof MG1655ΔlacItransformed with pET28a+arg1

  • 200μLof MG1655ΔlacItransformed with pET28a+arg1

Next Steps:

• Redo Calibration #2: Particle Standard Curve - Microsphere Protocol

• Overnight culture of 2B (2 colonies) and 2D (2 colonies) in DH5α

• If pET28a+arg1 transformation works

  • 2 MG1655ΔlacI overnight cultures (LB+KAN+IPTG)

  • 2 MG1655ΔlacI overnight cultures (LB+KAN)

  • streak a single MG1655ΔlacIcolony on an LB+KAN plate

Day 1, Monday 18/06

People In Lab: Amalia, Daniel, Jasmeen, Nina, Tashi

 

Agenda:

• LB+CAM plates
• Overnight culture of 2B (2 colonies) and 2D (2 colonies) (DH5α) for interlab
• 2 overnight cultures of LB+KAN+MG1655ΔlacI+IPTG
• 2 overnight cultures of LB+KAN+MG1655ΔlacI
• Streak plate pET28a+arg1 transformation on LB+KAN to get single colonies (MG1655ΔlacI)
• Autoclave microcentrifuge tubes

 

Equipment used:

• Autoclave
• Bunsen burner
• Shaker in room 315

 

Protocol: iGEM protocols used for LB+agar

 

Overnight cultures of colonies 2B+2D for interlab CFU

-4 LB+CAM tubes each with 5mL LB+5μL CAM

-2 colonies taken from 2B plate and inoculated

-2 colonies taken from 2D plate and inoculated

-all tubes placed in the shaker at 37°C at 250rpm

 

Serial dilution of IPTG

-dilution 1: 30μL of 1M IPTG stock in 10mL of LB = conc. 3mM

-10μL of dilution 1 in 10mL of LB = conc. 3μM

 

Overnight cultures of pET28a+arg1

-4 tubes

-2 with 5mL LB+IPTG+25μL KAN

-2 with 5mL LB+25μL KAN

-all tubes placed in the shaker at 37°C at 250rpm

 

Observations:

pET28a+arg1 transformation into MG1655ΔlacI

-No colonies on negative control plate or 100μL plate of transformed cells

-2 blobs on the 200μL plate of transformed cells

 

pET28a+arg1 transformation into MG1655ΔlacI negative control - 40μL untransformed cells

pET28a+arg1 transformation into MG1655ΔlacI 100μL transformed cells

pET28a+arg1 transformation into MG1655ΔlacI 200μL transformed cells

CFU colony count

		Colony 1				Colony 2
2B	dilution 3	210	201	224		134	0	300
	dilution 4	*	*	*		*	*	*
	dilution 5	*	*	*		*	*	*
2D	dilution 3	144	174	108		352	273	372
	dilution 4	31	16	33		20	1	11
	dilution 5	3	1	3		1	4	3

*plates were cracked because they were frozen

 

Next Steps:

• Redo interlab CFU protocol
• Let overnight cultures settle on bench (LB+KAN+MG1655ΔlacI+pET28a+arg1+IPTG) and (LB+KAN+MG1655ΔlacI+pET28a+arg1)
• Centrifuge pET28a+arg1 overnight cultures at 350g for 4 hours (LB+KAN+MG1655ΔlacI+pET28a+arg1+IPTG) and (LB+KAN+MG1655ΔlacI+pET28a+arg1)
• Streak out a single colony from the MG1655ΔlacI+pET28a+arg1 plate on an LB+KAN plate

 

Day 2, Tuesday 19/06

People In Lab: Amalia, Daniel, Nina, Tashi

 

Agenda:

• Continue interlab CFU protocol (up until plating)
• Centrifuge 1 overnight culture (LB+KAN+MG1655ΔlacI+IPTG) and 1 overnight culture (LB+KAN+MG1655ΔlacI) for 4 hours at 350g
• Keep 1 overnight culture (LB+KAN+MG1655ΔlacI+IPTG) and 1 overnight culture (LB+KAN+MG1655ΔlacI) on bench and observe
• Re-streak MG1655ΔlacI+pET28a+arg1 transformation on LB+KAN to get single colonies (was not incubated yesterday)
• Make new overnight culture of MG1655ΔlacI+pET28a+arg1

 

Equipment used:

• Bunsen burner
• Plate reader
• Shaker in autoclave room
• Centrifuge in room 301
• 37°C incubator in room 301

 

Protocol: Interlab protocol used for CFU experiement

 

Overnight culture of LB+KAN+MG1655ΔlacI+pET28a+arg1

-5mL LB+25μL KAN+incoulation from a blob from the LB+KAN+MG1655ΔlacI+pET28a+arg1 transformation plate

-tube placed in shaker at 37°C at 220rpm

 

Observations:

pET28a+arg1 induction

LB+KAN+MG1655ΔlacI+pET28a+arg1, LB+KAN+MG1655ΔlacI+pET28a+arg1+IPTG in centrifuge - cells pelleted at the bottom

-potential reason - too many gs

LB+KAN+MG1655ΔlacI+pET28a+arg1, LB+KAN+MG1655ΔlacI+pET28a+arg1+IPTG on bench - no separation

-potential reasons -arg1 not induced by IPTG?

-too hard flotation to see because there is no reporter

 

Centrifuged LB+KAN+MG1655ΔlacI+pET28a+arg1+IPTG (left )and LB+KAN+MG1655ΔlacI+pET28a+arg1 (right)

 

Next Steps:

• LB media
• LB+KAN plates
• Dilute overnight culture to OD=0.3
• Make IPTG dilution
• Induce LB+KAN+MG1655ΔlacI+pET28a+arg1 culture with 3μM IPTG for 22 hours (2 tubes)
• Make new LB+KAN+MG1655ΔlacI+pET28a+arg1 overnight culture for 22 hours (2 tubes)
• Count colonies for interlab CFU
• Streak out a single colony from the (MG1655ΔlacI) plate on an LB+KAN plate
• Make protocol for growth curve for dry lab

 

Day 3, Wednesday 20/06

People In Lab: Amalia, Daniel, Jasmeen, Nina, Tashi

 

Agenda:

• Make LB liquid media
• Make LB+KAN plates
• Count colonies for interlab CFU
• Streak out a single colony from the MG1655ΔlacI+pET28a+arg1 plate on an LB+KAN plate
• Induce LB+KAN+MG1655ΔlacI+pET28a+arg1 culture with 3μM IPTG for 22 hours (2 tubes)
• Make new LB+KAN+MG1655ΔlacI+pET28a+arg1 overnight culture for 22 hours (2 tubes)

 

Equipment used:

• Autoclave
• Bunsen burner
• Shaker in autoclave room
• 37°C incubator in room 301
• Spectrophotomter

 

Protocol: iGEM protocols used for LB+agar, interlab protocol used for CFU experiement

 

IPTG dilution #1 in LB

C₁V₁ = C₂V₂

C₁ = 1M

C₂ = 3mM

V₂ = 10mL

V₁ = 30μL

= take 30ul of stock and add to 10mL LB+KAN

 

IPTG dilution #2 in LB

C₁V₁ = C₂V₂

C₁ = 3mM

C₂ = 3uM

V₂ = 25mL

V₁ = 25μL

= take 25ul of dilution #1 and add to LB+KAN

 

pET28a+arg1 overnight culture

1:8 dilution, measure OD, OD = 0.25

0.25x8 = 2

C₁V₁ = C₂V₂

C₁ = 2

C₂ = 0.3

V₂ = 25mL

V₁ = 3.75mL

 

Total volume = 25mL

 

25mL - 3.75mL = 21.25mL

-21.25mL LB + KAN + 3.75mL overnight culture

-21.25mL LB + KAN + IPTG + 3.75mL overnight culture

 

Abs₆₀₀ blank (LB+KAN+IPTG) = 0.00 (tared)

Abs₆₀₀ (LB+KAN+IPTG+ARG1) = 0.127

 

Both tubes placed in shaker at 37°C at 220 rpm (one with IPTG and one without)

 

Observations:

CFU colony count #2

		Colony 1				Colony 2
2B	dilution 3	180	138	40		77	78	91
	dilution 4	6	16	8		10	8	4
	dilution 5	2	3	2		1	0	1
2D	dilution 3	215	150	149		83	115	274
	dilution 4	45	28	28		30	14	28
	dilution 5	2	2	2		0	4	4

 

Colony forming unit calculation (using Dilution 3 data)

Calculated for Interlab Form IV

(# of colonies) x (Final Dilution Factor) = CFU/mL

Final Dilution Factor = 8 x 10⁴

 

	Colony 1	Colony 2	-		Colony 1	Colony 2
2B	8.64 x 10^6	6.16 x 10^6	-	2D	1.72 x 10^7	6.64 x 10^6
	1.104 x 10^7	6.24 x 10^6	-		1.2 x 10^7	9.20 x 10^6
	3.20 x10^6	7.28 x 10^6	-		1.192 x 10^7	2.192 x 10^7

MG1655ΔlacI+pET28a+arg1 streak to get single colonies

 

Next Steps:

• LB+KAN plates
• Make KAN dilutions
• Order primers
• Order blue protein

 

Day 4, Thursday 21/06

People In Lab: Amalia, Daniel, Jasmeen, Nina

 

Agenda:

• Make LB + KAN plates
• Make KAN dilutions
• Make overnight culture of LB+KAN+MG1655ΔlacI+pET28a+arg1 for dry lab growth curve
• Order primers for pET28a+arg1
• Order blue protein

 

Equipment used:

• Bunsen burner
• Shaker in room 315

 

Protocol:

KAN dilutions

(800μL of nuclease-free water+200μL 50mg/mL KAN = 1mL 10mg/mL KAN) x3

 

Overnight culture of LB+KAN+MG1655ΔlacI+pET28a+arg1 for growth curve

-(15mL LB + 75μL KAN + incoulation from colony on newest MG1655ΔlacI+pET28a+arg1 plate) x3

-tube placed in shaker at 37°C at 220rpm

 

Observations:

MG1655ΔlacI+pET28a+arg1 streak to get clonal colonies

 

Next Steps:

• LB+KAN plates
• Make KAN dilutions
• Do digest and run gel
• Dry lab growth curve
• Order amilCP

 

Day 5, Friday 22/06

People In Lab: Amalia, Nina, Daniel, Tashi

 

Agenda:

• Make LB + KAN plates
• Make KAN dilutions
• LB+KAN+MG1655ΔlacI+pET28a+arg1 growth curve
• Order amilCP

 

Equipment used:

• Bunsen burner
• Shaker in autoclave room
• Plate reader
• Autoclave

 

Protocol: iGEM protocols for LB+agar and KAN dilutions

 

KAN dilutions

-80mL of 10mg/mL were made

-50 microcentrifuge tubes were filled with 1mL each and 30mL remains in a falcon tube in the fridge

 

LB+KAN+MG1655ΔlacI+pET28a+arg1 growth curve

-1:8 dilution made from each overnight culture (175mL LB+KAN and 25mL culture in well)

-200μl LB+KAN pipetted in well (blank)

-measure OD₆₀₀

-subtract blank value, multiply by 8 to get original concentration (OD)

-dilute each overnight culture to get an OD of 0.1

-take OD again (0h timepoint)

-place the three replicates back in the shaker and measure again every hour (200μL)

 

Observations:

Volumes for LB+KAN+MG1655ΔlacI+pET28a+arg1 in growth curve cultures based on OD values of 1:8 dilutions-blank value multiplied by 8

Overnight culture 1 1:8 dilution OD without blank = 0.2209

Overnight culture 1 original OD = 1.3576

Replicate 1 LB+KAN = 23.158mL

Replicate 1 overnight culture = 1.841mL

 

Overnight culture 2 1:8 dilution OD without blank = 0.2167

Overnight culture 2 original OD = 1.324

Replicate 2 LB+KAN = 23.111mL

Replicate 2 overnight culture = 1.888mL

 

Overnight culture 3 1:8 dilution OD without blank = 0.2235

Overnight culture 3 original OD = 1.3784

Replicate 3 LB+KAN = 23.186mL

Replicate 3 overnight culture = 1.814mL

 

LB+KAN+MG1655ΔlacI+pET28a+arg1 in growth curve data

0	0.094	0.0942	0.1022	*0.0512
1	0.1808	0.1757	0.1713
2	0.255	0.2538	*0.4448
3	0.3391	0.3247	0.3332
4	0.3937	0.4075	0.4032
5	0.4539	0.4688	0.4326
6	0.4652	0.4797	0.4705

*Replicate 3 OD at 2h has an incorrect measurement. All 2h replicates were retested an hour later and they all had very similar values.

*All measurements shown already have the blank value subtracted from the raw value

 

Next Steps:

• PCR pET28a+arg1 with new primers
• Give Scott LB plate to streak with BL21
• LB+AMP plates (for gibson positive control)
• Order gibson
• Order amilCP