Overnight culture of MACH1 and DH5α E. coli
Inoculate MACH1 and DH5α E. coli from glycerol stock into 5 ml LB broth. Incubate at 37 ^◦C with 150 rpm shaking overnight.

Prepare CCMB80 competent cell buffer (100ml)

Components	Amount	Final concentration
1M pH 7.0 CH₃COOK stock solution	1 mL	10 mM
CaCl₂.2H₂O	1.18 g	80 mM
MnCl₂.4H₂O	0.4 g	20 mM
MgCl₂.6H₂O	0.2 g	10 mM
Glycerol	10 mL	10 % v/v
MiliQ H₂O	added to 100 ml	Final concentration

Adjust pH to 6.4 by 0.01M HCl. Sterile by filtration through 0.22 μm filter. Keep in 4 ^◦C

Nat

Competent cell preparation 2 – 20/06/18

Protocol adjusted from iGEM Competent Cell Production Protocol (http://parts.igem.org/Help:Protocols/Competent_Cells)

Inoculate 1 ml of MACH1 and DH5α E. coli O/N culture in 100 ml of fresh LB broth. Incubate at room temperature, with 50 rpm shaking until OD600 reach 0.3 - 0.5 (3.30 hours).
Chill the culture on ice for 30 min.
Centrifuge at 3000 g for 10 min. Discard supernatant.
Resuspend cell pellet in 50 ml ice cold sterile H₂0.
Centrifuge at 3000 g for 10 min. Discard supernatant.
Resuspend cell pellet gently in 20 ml ice cold CCMB80 buffer.
Centrifuge at 3000 g for 10 min. Discard supernatant.
Resuspend cell pellet gently in 8 ml ice cold CCMB80 buffer.
Aliquot 50 𝜇L to cold 2 ml Eppendorf tube. Snap freeze in liquid N₂.
Store in -80 ^◦C

Nat

Test for competency – 21/06/18

Test for competency according to iGEM Competent Cell Test Kit protocol (http://parts.igem.org/Help:2017_Competent_Cell_Test_Kit)

Result: DH5α 1.5 x 10⁷ CFU/μg

MACH1 5 x 10⁶CFU/μg

Nat

Week 28

Culture SIEC and SIECΔp1 E. coli – 11/07/18

SIEC and SIECΔp1 E. coli strain arrived from Spain.
Streak SIEC and SIECΔp1 E. coli from agar stab stock on LB agar plate.

Nat

Culture SIEC and SIECΔp1 E. coli –12/07/18

Pick a single colony from SIEC and SIECΔp1 into 5 ml LB broth. Incubate at 37 ^◦C with 150 rpm shaking overnight.

Nat

Prepare SIEC and SIECΔp1 competent cell stocks – 13/07/18

Prepare competent cell stocks from SIEC and SIECΔp1 using the same protocol mentioned in date 19/06/18 and 20/06/18.

Nat

Week 29

pBAD and pTac Transformation – 16/07/18

Transform pBAD (BBa_K228005) and pTac (BBa_731500) into MACH1 E. coli according to iGEM transformation protocol (http://parts.igem.org/Help:Protocols/Transformation).

Primers arrived today. Dilute primer into 100 μM stock and 10 μM working stock.

Nat

PCR reporter genes and T3SS signal – 17/07/18

PCR amplify reporter genes sfGFP (BBa_I746916), NanoLuc (BBa_K1159001), mCherry (BBa_J06504 ), and beta-lactamase (BBa_K1189008) from Biobrick distribution kit.

PCR assemble T3SS signal from 5 overlapping oligonucleotides. This signal encode 20 amino acid from N-terminal Map genes.

Template	Primer 1	Primer 2	Product	Size (bp)
1. NanoLuc BBa_K1159001	Nluc-F	Nluc-R	NanoLuc	555
2. sfGFP BBa_I746916	GFP-F	GFP-R	sfGFP	748
3. mCherry BBa_J06504	mCH-F	mCh-R	mCherry	740
4. Beta-lactamase BBa_K1189008	Blac-F	Blac-R	Beta-lactamase	822
5. Map (no template)	Map20-1, Map20-2, Map20-3, Map20-F2, Map20-R2 (55)		Map20	136
6. SIEC E. coli genomic DNA	EspD-UF	EspD-UR	EspD	1180

Pfu-X7 PCR reaction components

Components	50 µl reaction	Final conc.
10X home-made X7 buffer	5 µl	1X
dNTP mixture (2.5 mM)	4 µl	200 µM
Pfu-X7 polymerase (2U/µl)	0.5 µl	1 Unit/50 µl
Forward primer (10 µM)	2.5 µl	0.5 µM
Reverse primer (10 µM)	2.5 µl	0.5 µM
Template	1 µl	<250ng DNA
MiliQ H₂O	To 50 µl	-

Pfu-X7 PCR temperature profile

Step	Temperature	time	Cycle
Initial denaturation	98 ^◦C	30 sec	-
Denaturation	98 ^◦C	10 sec	35 Cycles
Annealing	55 ^◦C	15 sec
Extension	72 ^◦C	45 sec
Final Extension	72 ^◦C	5 min	-
Hold	16 ^◦C	∞	-

Gel result

alt_text

1. NanoLuc Fail
2. sfGFP Fail
3. mCherry Fail
4. Beta-lactamase Fail
5. Map20 (T3SS signal) Success

6. EspD Success

The failed PCR may result from incompatible PCR polymerase enzyme or buffer. Next time change PCR enzyme to Q5 DNA polymerase.

Nat, Selma

PCR reporter genes – 18/07/18

PCR amplify reporter genes sfGFP (BBa_I746916), NanoLuc (BBa_K1159001), mCherry (BBa_J06504 ), and beta-lactamase (BBa_K1189008) again using Q5 DNA polymerase.

Template	Primer 1 (Tm)	Primer 2 (Tm)	Product	Size(bp)
1. sfGFP BBa_I746916	GFP-F (61)	GFP-R (55)	sfGFP	748
2. NanoLuc BBa_K1159001	Nluc-F (68)	Nluc-R (60)	NanoLuc	555
3. mCherry BBa_J06504	mCH-F (63)	mCh-R (58)	mCherry	740
4. Beta-lactamase BBa_K1189008	Blac-F (61)	Blac-R (58)	Beta-lactamase	822

Second PCR using PCR product from the first PCR as a template.

Template	Primer 1	Primer 2	Product	Size(bp)
1. sfGFP	GFP-F	GS-His-R	sfGFP-His	772
2. NanoLuc	Nluc-F	GS-His-R	NanoLuc-His	579
3. mCherry	mCH-F	GS-His-R	mCherry-His	762
4. Beta-lactamase	Blac-F	GS-His-R	Beta-lactamase-His	846

Q5 PCR reaction components

Components	50 µl reaction	Final concentration
Q5 High-Fidelity 2X Master Mix	25 µl	1X
Forward primer (10 µM)	2.5 µl	0.5 µM
Reverse primer (10 µM)	2.5 µl	0.5 µM
Template	1 µl	<250ng DNA
MiliQ H₂O	To 50 µl	-

Q5 PCR temperature profile

Step	Temperature	time	Cycle
Initial denaturation	98 ^◦C	30 sec	-
Denaturation	98 ^◦C	15 sec	35 Cycles
Annealing	55 ^◦C	25 sec
Extension	72 ^◦C	45 sec
Final Extension	72 ^◦C	5 min	-
Hold	16 ^◦C	∞	-

Gel result

alt_text

1. sfGFP-His Success

2. NanoLuc-His Success
3. mCherry-His Success
4. Beta-lactamase-His Success

Purify sfGFP-His, NanoLuc-His, mCherry-His, Beta-lactamase-His, EspD, and Map20 PCR product using E.Z.N.A.® Cycle Pure Kit.

Nat, Eric

Restriction enzyme digestion – 19/07/18

Digestion

DNA	Restriction enzyme 1	Restriction enzyme 2
pBAD	SpeI	PstI
Map20	XbaI	AgeI
mCherry	XmaI	PstI
sfGFP	XmaI	PstI
Beta-lactamase	XmaI	PstI
NanoLuc	XmaI	PstI

25 μl restriction enzyme digestion reaction components

10X Cutsmart™ buffer 2.5 μl

DNA 250 ng

20U/μl Restriction enzyme 0.5 μl

H₂0 to 25 μl

Temperature profile

37 C 30 min

80 C 20 min

Digestion products are purified by E.Z.N.A.® Cycle Pure Kit.

Selma, Eric

Ligation and Cloning – 13/07/18

Week 28

Cloning biobricks – 19/09/18

PCR products from the XX.09.18 and the plasmid backbone were digested with EcoRI and PstI in the following reactions:

pSBIC3
Plasmid	50 uL
EcoRI	1 uL
PstI	1 uL
10x Cutsmart Buffer	10 uL
ddH2O	38 uL

Map20, CesF and CesT
PCR product	5 uL
EcoRI	0,2 uL
PstI	0,2 uL
10x Cutsmart Buffer	1 uL
ddH2O	3,6 uL

Map20-mCherry
PCR product	7 uL
EcoRI	0,2 uL
PstI	0,2 uL
10x Cutsmart Buffer	1 uL
ddH2O	1,6 uL

Program:

37 oC 90 min

85 oC 20 min

16 oC inf.

Ligation reaction between digested backbone and digested PCR products.

Ligation reactions
Digested pSBIC3	1 uL
Digested insert (Map20, Map20-mCherry, CesT, CesF)	1 uL
2x Rapid ligase buffer	2,5 uL
T4 ligase	0,5 uL

The mixtures were incubated at 16 oC for xx minutes.

Transformation of ligation products into Mach1 cells.

Transformation as in TRANSFORMATION PROTOCOL until step 14 with 2 uL ligation reaction and 50 uL competent Mach1 cells.

Selma

Making BioBricks

Liposome charaterization

Protoplast experiment with plants

Week 36

Preparation of pilot experiment – 06/09/18

Picked colonies from plates with the relevant strains and started an overnight liquid culture. Note: The colonies picked were from chose three SIEC strains, one containing GFP, another mCherry and then Beta-Lactamase to serve as a negative control.
Looked at the protocol which the 2011 Hokkaido iGEM team used for their onion experiment and adjusted it to our strains.

Stephanie and Eiríkur

Further preparation for pilot experiment – 07/09/18

Started a new overnight culture of cells (from the 6th) in induction media containing IPTG and Arabinose.
Prepared 1L of MgM-MES buffer pH5.0 using the following recipie:

MgM-MES buffer pH5.0:

170mM MES

5mM KCl

75mM (NH4)2SO4

0.5mM K2SO4

8uM MgCl2

38mM Glycerol

0.1% Casaminoacids

Fill up to 1L of water and autoclave to sterilize.

Stephanie and Eiríkur

Week 37

Preparation of pilot experiment – 10/09/18

Due to a delay because of missing mannitol, two new overnight cultures were started with the strains mentioned above. The two cultures were made differently to see if there is a difference in time of the Induction of Injectisome production.

Culture 1(mCherry,GFP, beta-lactamase strains): Prepared using LB media with Chloramphinicol and 1%arabinose(to induce the production of all the reporter proteins).

Culture 2:(mCherry,GFP, beta-lactamase strains): Prepared using LB media with Chloramphinicol ,1%arabinose(to induce the production of all the reporter proteins) and 0.1mM IPTG(To induce the production of the injectisome)

Stephanie and Eiríkur

Pilot experiment – 11/09/18

Decision was made to use D-Sorbitol instead of Manitole

The overnight cultured cells were spun down at 3000rpm, for 10 min at 35C. Supernatant removed.
5mL MgM-MES (pH5.0), Chloramphenicol, L-arabinose and IPTG were added to the bacterial pellet and resuspended by vortexing.cells were culteured at 37C, shaking at 200rpm for 4 hours.
While cultures were incubating, onion sheets were prepared using this protocol
When cultures had incubated we spun down the culture at 3000rpm, for 10 min at 25C, supernatant was removed
Pellet was resuspended with 2 ml of MgM-MES (pH5.0) containing 0.4 M Mannitol and spun down at 3000rpm, for 10 min at 25C, supernatant was removed. This was repeated 3 times.
Pellet was resuspended with 2 ml of MgM-MES (pH5.0) containing 0.4 M Sorbitol, chloramphenicol, L-arabinose and IPTG.
Light absorbance was measured at 600nm using a spectrophotometer and the concentration of cells was adjusted to ΔOD600 = 0.06 by diluting with the same medium mentioned above.
To infect the onion cells 500ul of the culture fluid was put onto the pre-treatd onion cell-sheets. The whole thing was left at room temperature. We made two samples for each culture, one was left for 2 hours the other one was left overnight

Cells were observed under a fluorescent microscope

Stephanie and Eiríkur

Onion preparation– 25/09/18

7 Fresh epidermal sheets of onion cells were cut out and put on microscope-slides

The onion cell sheets were attached to the microscope slide with agarose

A solution containing 1% Cellulase, 0.1%Pectolyase, 0.4M Sorbiltole-D and MgM-MES buffer, was put onto the onion sheets

1 sample was a control with no solution, the rest(6) were observed after 10min, 20min and 45min(duplicates were made for each time interval), under a microscope to see how the onion cells changed according to the incubation time of the enzymes

t=0

T=20

t=45

There were no conclusive results, might be because the incubation time was too short, the buffers were old or the enzymes did not work as suspected

Further preparation of onion sheets and tobacco plant observations– 27/09/18

New buffers and solutions were made:

1 liter MgM-MES buffer,
500ml 0.4M Sorbitol-D solution in MgM-MES buffer,
50ml of 1%Cellulase and 0.2% pectolyase in a 0.4M Sorbitol-D solution in MgM-MES
- 7 Fresh epidermal sheets of onion cells were cut out and put on microscope-slides

The onion cell sheets were attached to the microscope slide with agarose

A solution containing 1% Cellulase, 0.1%Pectolyase, 0.4M Sorbiltol-D and MgM-MES buffer, was put onto the onion sheets

1 sample was a control with no solution, the rest(6) were observed after 10min, 20min and 60min(duplicates were made for each time interval), under a microscope to see how the onion cells changed according to the incubation time of the enzymes

A single tobacco plant was obtained for the purpose of trying to observe protoplasts
The cuticule was scraped off as best as possible
A solution containing 1% Cellulase, 0.1%Pectolyase, 0.4M Sorbiltol-D and MgM-MES buffer, was put into a petri dish, the piece of leaf without it´s cuticle was put on top of the solution and left there for an hour
The treatment was done on a big leaf from the plant, a smaller leaf, and a tiny leaf, to see of there was any visible difference in amount of protoplasts
The leafs and its solution were observed under through a microscope which revealed burst cells and very few protoplast

Osmolarity test on onion sheets and tobacco plant – 02/10/18

We had been using 0.4M solution of Sorbitol through the entire experiment so we decided to test different concentrations, to see if it made any difference. We decided furthermore to switch from Sorbitol to Mannitol since a new batch of mannitol just arrived

A 12 well culture plate was obtained. Each well was filled with a solution containing MgM-MES buffer, 0.1% pectolyase, 1%cellulase and Mannitol in 9 different concentrations ranging from 0.4M-0.8M.
The cuticle of the tobacco leaves were scraped of and but downside-down into each of the wells
The first 9 wells contained 0.4;0.45;0.5;0.55;0.6;0.65;0.7;0.75 and 0.8 M Mannitol,respectively, plus the MgM-MES and enzymes. The last three contained onion cell sheets in 0.6,0.7 and 0.8 M Mannitol with the solution mentioned above.
Samples were taken after 3 hours and looked at under a fluorescent microscope.
We saw tobacco protoplasts in most samples but the sample with the highest concentration protoplast appeared to be the one containing 0.6-0.65 M of Mannitol
From looking at the onion cells, they appeared to be best treated with a 0.8M of Mannitol
We decided to move forward with 0.6M Mannitol for the tobacco leaves and 0.8M for the Onion cells

Onion and tobacco assay – 05/10/18

8/9/18 - Stephanie & Eirikur

Onion assay pilot experiment:

Delayed due to missing Mannitol

Spin down the cultured cells at 3000rpm, for 10 min at 35C. Remove supernatant.
Add 5mL MgM-MES (pH5.0), Chloramphenicol, L-arabinose and IPTG to the bacterial pellet. Resuspend it using vortex mixer. Culture cells at 37C, shaking at 200rpm for 4 hours. This step is required because genes encoding salmonella T3SS are expressed in acidic environment.
While cultures are incubating, prepare onion sheets:
Cut an onion into four pieces.
Take out a fresh sheets of epidermal cells (second or third layer from the surface of onion).
Cut the layer into rectangular shape (about 1.5cm x 3cm)
Tear off a cell sheet from the inner surface of the layer.
Put the sheet onto a slide glass at back face up because the upper face deflects enzyme solution, and fix the edge of the sheet onto slide glass with 1% agarose gel.
Fill a petri dish with 1% cellulase and 0.1% pectolyase in 0.4 M Mannitol solution (pH7.0), then submerge the cell sheet fixed on slide glass into the enzyme solution. Incubate for 8 min at RT.
Prepare 3 petri dishes filled with MgM-MES pH5.0 containing 0.4 M Mannitol. After processing, wash cells stepwise soaking in these petri dishes to remove enzyme solution.
Remove excess moisture, and transfer the sample to another petri dish for infection.
Spin down the culture fluid at 3000rpm, for 10 min at 25C. Remove supernatant.
Resuspend the pellet with 2 ml of MgM-MES (pH5.0) containing 0.4 M Mannitol, then spin down the culture fluid at 3000rpm, for 10 min at 25C. Remove supernatant. Repeat this step 3 times. (This is done to remove the toxic substances that are produced by E. coli and adjust the osmotic pressure to that in onion cells)
Resuspend the pellet with 2 ml of MgM-MES (pH5.0) containing 0.4 M Mannitol, Tetracycline, chloramphenicol and L-arabinose.
Measure the light absorbance at 600 nm using a spectrophotometer, then adjust the concentration of cells to ΔOD600 = 0.06 by diluting with the same medium mentioned above.
To infect onion cells with E. coli add 500 ul of the culture fluid onto pre-treated onion cell-sheets. Leave at RT in petri dish (Prevent drying)
Remove the bacterial culture fluid carefully by micropipette so that the cell-sheets are not to be torn. Observe the cells under a fluorescencent microscope.

Week 41

Remaking protoplast and preparing bacteria– 10/10/18

We needed to remake protoplast in 5,0 pH and induce older bacteria samples to mix and check for secretion.

Victoria

Collecting protoplasts made 09/10/18

Identify which well contains protoplasts for onion and tobacco samples with a microscope
Add 2 ml sample to an eppendorf tube
Spin down samples at low tempo for 3 min
Remove supernatant
Flick tubes to mix
Check new samples for protoplast using a microscope
Steps 1-6 were repeated 2x, with batch number 1 containing the most protoplasts

Inducing bacteria

Samples

mCherry: I-S-C-, I+S+C+

GFP: I-S-C-, I+S+C+

EspD:

bacteria (older sample from the fridge, since no new overnight culture was started beforehand) was spun down and pellet resuspended in 5 ml fresh LB+CaM media
400 microliter resuspended culture added to 10 ml LB+CaM + 500 microliter 20 % Arabinose + 2mM IPTG to induce bacteria
Incubation
1. 1st batch start: 12:53
2. 2nd batch start: 13:20

Remaking onion and tobacco protoplasts at pH 5,0

Prepared 500 ml of MgM-MES buffer pH5.0 using the following recipe:

MgM-MES buffer pH5.0:

170mM MES: 19,66 g

5mM KCl: 0,19 g

75mM (NH4)2SO4: 4,96 g

0.5mM K2SO4: 0,044 g

8uM MgCl2: 0,068 g

38mM Glycerol: 1,4 ml

0.1% Casaminoacids: 0,5 g

pH was adjusted with 5,0 M KOH
Dilute buffer to 500 ml
0,8 M and 0,6 M Mannitole buffer was prepared (250 ml 0,6 M Mannitole = 27,5, 250 ml 0,6 M Mannitole = 36,5 g )
Peel inner epidermis from onion and tobacco
Leave in Mannitole buffer for min 4. hours
Mix bacteria and protoplasts
Incubate overnight

Week 41

Washing protoplasts to remove bacteria – 11/10/18

We needed to separate bacteria from protoplasts to use a fluorescent microscope to check for secretion into protoplasts. It turned out washing by spinning down protoplasts, removing supernatant containing bacteria and resuspending pellet was an inefficient way to do this.

Victoria

Note: Only the onion protoplast samples were used

Mix samples gently by flicking
Centrifuge for 3 min at 150 RCF (or until pellet appear)
Remove supernatant
Resuspend pellet in 200 microliter 0,8 M mannitole buffer
1. Check samples in microrscope to ensure protoplast are stable and kept in sample
2. All samples were washed 6 times by following step 1-4
  1. Note: This wasn't sufficient to remove bacteria entirely
  2. Note: Protoplast seemed to shrink during the time of the experiment
  3. Check samples in fluorescent microscope

Using Lyzozyme to remove bacteria – 11/10/18

We wanted to see if digesting the bacteria would be more efficient than washing the samples. This experiment showed that separating protoplast from bacteria was innificient.

Note: This experiment used the tobacco samples left over from the day before

Victoria

Spin tobacco 3 min 150 RCF
Remove supernatant
Add 200 microliter of lyzozyme solution to samples
1. Lyzozyme solution: 10 g Lysozyme from chicken egg white,1 ml Mannitole buffer
2. Check samples every 30 min in microscope
  1. Note: Bacteria did not get removed

Leakiness characterization

Week 36

Overnight of strains (1) – 06/09/18

An overnight of the strains XXX was made.

Nat

Induction of strains + sample taking over time (1) – 07/09/18

The first experiment in relation to the examination of the injectisome leakiness was conducted with the strains from the overnight. We followed this (INSERT) protocol.

The bacteria were induced with IPTG and arabinose and a sample was taking at T=0 for each strain. Thereafter a sample was taken every 2 hours. The samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

Attila, Nat and Lasse

Induction of strains + sample taking over time (1) – 08/09/18

The last sample of this experiment, T=overnight, was taken for each strain. The samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

Attila, Nat and Lasse

Week 37

Overnight of strains (2) – 13/09/18

An overnight of the strains XXX was made.

Nat

Induction of strains + sample taking over time (2) – 14/09/18

The second experiment in relation to the examination of the injectisome leakiness was conducted with the strains from the overnight. We followed this (INSERT) protocol.

The bacteria were induced with IPTG and arabinose and a sample was taking at T=day0 for each strain. Thereafter the bacteria were placed at 16 ^oC. The T=day0 samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

Attila, Nat and Lasse

Induction of strains + sample taking over time (2) – 15/09/18

A sample, T=day1, was taken from the tubes incubated at 16 C^O for each strain. The T=day1 samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

Attila, Nat and Lasse

Induction of strains + sample taking over time (2) – 16/09/18

A sample, T=day2, was taken from the tubes incubated at 16 C^O for each strain. The T=day2 samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

Attila, Nat and Lasse

Induction of strains + sample taking over time (2) – 17/09/18

A sample, T=day3, was taken from the tubes incubated at 16 C^O for each strain. The T=day3 samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

Attila, Nat and Lasse

Induction of strains + sample taking over time (2) – 18/09/18

A sample, T=day4, was taken from the tubes incubated at 16 C^O for each strain. The T=day4 samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

Attila, Nat and Lasse

Induction of strains + sample taking over time (2) – 19/09/18

A sample, T=day5, was taken from the tubes incubated at 16 C^O for each strain. The T=day5 samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

Attila, Nat and Lasse

Induction of strains + sample taking over time (2) – 20/09/18

A sample, T=day6, was taken from the tubes incubated at 16 C^O for each strain. The T=day6 samples were centrifuged and the supernatant transferred to another tube. The tubes with the pellet and the tubes with the supernatant were placed in the freezer.

Attila, Nat and Lasse

Measurement of fluorescence of supernatant and pellet (2) – 21/09/18

The fluorescence was measured of the supernatant and the pellet, for the experiment where samples were taken once a day. The supernatant was measured directly whereas the pellet was first resuspended and diluted 10-fold using LB+CAM media.

Experiments with supported lipid bilayers

Week 28

Learning to make liposomes – 12/07/18

We were introduced to the process of making liposomes by the nice people in Nikos Hatzakis lab, Søren Schmidt-Rasmussen Nielsen and Mette Galsgaard Malle who gave us the Liposome preparation protocol. We made some tryout liposomes, that were flashed freezed and stored in -20 °C.

Selma, Sofia and Attila

Week 34

Transforming SIEC strains with GFP plasmids – 24/08/18

To visualize our bacteria in the microscope together with a membrane we needed it transformed with a GFP plasmid. We used plasmids from the distribution kit that functioned as Test Device 1 (BBa_J36400) and positive control (BBa_I20270) in the InterLab study.

Transformation as described in Transformation protocol until step 14, the following combinations of strain and plasmid was made:

SIEC x BBa_I20270
SIECΔp1 x BBa_I20270

Plates were made from agar and LB with chloramphenicol (34 μg/mL) (CAM). Plates with bacteria spent 17 hours in the 37 °C incubator before being kept in fridge.

Selma

Week 35

Testing transformed bacteria for fluorescence – 27/08/18

To test if the transformation was successful fluorescence were measured on a plate reader.

Colonies from transformation 24/08/18 was picked into 50 mL falcon tubes with 5 mL LB media (that also contained 1% arabinose). Incubated at 37 °C at 130 rpm for 6 hours. Triplicates of each 100 μL of culture was pipetted into wells of a 96 well-plate. Fluorescence and absorbance was measured. LB media was used as blank.

Glycerol stocks were made from the cultures.

Selma

Making lipid film stocks 1 – 29/08/18

In order to make the liposomes for later experiments quickly and to ensure that the liposomes have the same composition, we made lipid film stocks from a lipid mastermix.

First, we weighed about 25 mg of powdered lipids.

Lipid	Mass (mg)
DOPC	24,8
DOPS	25,2
SM	25*
Cholesterol	25*

*lipids were obtained as powder in this amount from the manufacturer

Lipids were dissolved in 1 mL chloroform.

To properly store the lipids, we used nitrogen flow to exchange air in the glass vials with nitrogen. Because some of the solvent evaporated during preparation due to the nitrogen flow, the lipid film stocks had to be redone 31/08/18.

Second, we preparred biotin stock 2. The biotin stock concentration was 0.5 μg/μL (stock 1). 150 μL of biotin stock 1 was added to 600 μL chloroform to make biotin stock 2 (0.1 μg/μL).

Selma and Lasse

Microscope tryout – 30/08/18

We got an introduction to the microscope and saw our bacteria in the microscope for the first time. We used the confocal microscope in Associate Professor Nikos Hatzakis' laboratory. Introduction to the microscope was kindly performed by Søren Schmidt-Rasmussen.

Bacteria incubation

We added 20 μL of glycerol stock from the 27/08/18 to 5 mL LB media with CAM in 50 mL falcon tubes (SIEC and SIECΔp1 with GFP, BBa_I20270).

Put in incubator at 37°C at 130 rpm for 3 hours.

Preparation of supported lipid bilayer

Supported lipid bilayer was made as described in the Moran-Mirabel protocol, step 8 to 12. For this first tryout, we used the liposomes made 12/07/18 (not right membrane composition). Microscopy slides were washed with Tris buffer. Membrane contained the dye Atto655.

Microscopy

Microscope slides were washed with the same media as used for cultivation of the bacteria. 100 μL bacteria culture was added to a slide well. We used lasers of 488 nm and 635 for excitation. FRAP was used to see if the membrane was intact, which was not the case.

Selma, Attila, Lasse and Nat

Making lipid film stocks 2 – 31/08/18

We wanted to make a mixture containing sphingomyelin (SM), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), and cholesterol (Chl) was used for the supported lipid bilayer and liposomes in a ratio of 44:24:12:20 (SM:DOPC:DOPS:Chl).

Chloroform in stocks from 29/08/18 was evaporated with nitrogen flow. Masses of lipids left were weighed. 1 mL solvent was added to each lipid vial to make stock 1.

Lipid	Mass (mg)
DOPC	25,6*
DOPS	21
SM	24,4
Cholesterol	24,9

*This number is higher than 29/08/18. This means that the concentration for DOPC is not exact.

Dilutions of each lipid stock ( called "Stock 2") were made:

Lipid	Volume of Stock 1	Methanol	Chloroform
DOPC	39 μL**	0 μL	960 μL
DOPS	48 μL	476 μL	476 μL
SM	41 μL	480 μL	480μL
Cholesterol	40 μL	0 μL	960 μL
Atto655 (0.5 μg/μL)	20 μL	0 μL	980 μL

**If the mass had corresponded to what was measured 29/08/18 this would have been 40 μL instead of 39 μL.

Three mixtures were made from the stock 2.

Lipid, stock 2	Mix 1	Mix 2	Mix 3
DOPC (1μg/μL)	75 μL	75 μL	75 μL
DOPS (1 μg/μL)	39 μL	39 μL	39 μL
SM (1 μg/μL)	133 μL	134 μL	134 μL
Cholesterol (1 μg/μL)	31 μL	31 μL	31 μL
Atto655 (0.01 μg/μL)	27 μL	27 μL	0 μL
Biotin (0.1 μg/μL)	60 μL	0 μL	0 μL

Mixtures were aliquoted into glass vials to contain 0.2 μmol lipids:

	Mix 1	Mix 2	Mix 3
μL mastermix to reach 0,2 μmol	37 μL/vial	31 μL/vial	28 μL/vial

Vials were kept in -20°C.

Selma

Week 36

Making lipid film stocks 3 – 05/09/18

To finish the lipid film stocks, solvent was evaporated with nitrogen flow for about 5 minutes. Then vials were centrifuged on a ScanVac for 15 minutes under vacuum. Vials containing lipid films were stored at -20 °C.

Selma

Streaking bacteria – 05/09/18

Bacteria from glycerol stock 27/08/18 (transformations 24/08/18) were streaked on LB agar plates with CAM (34 μg/mL). Incubated overnight and kept in fridge.

Selma

Week 37

Full protocol tryout 1 – 12/09/18

We inoculated colonies from plates that were made the 05/09/18 in LB media with arabinose (1%), IPTG (0.1 mM) and CAM (34 μg/mL). The cultures were incubated for 2.5 hours at 37 °C 160 rpm. Meanwhile lipid films were rehydrated, extruded through a 200 nm filter and underwent 10 cycles of freezing in nitrogen and heating in a water bath, see Liposome preparation protocol. Liposomes were used to make a supported lipid bilayer on a microscope slide. The rest of the liposomes were kept in -80 °C freezer.

After 2.5 hours of incubation, we measured OD of the bacterial culture (results below) and pipetted 100 uL of bacteria culture onto each slide with bilayer. This was incubated at 37 °C in 3 hours.

After incubation, we looked at each slide in the microscope, taking pictures before and after washing with 100 uL media 3 times.

Sofia and Selma

InterLab

Throughout the interlab experiments, we followed the protocol provided by the measurement committee. The protocol can be seen here.

In the following notebook we have focused on what we have done and then linked to the interlab page on this wiki. We have chosen not to incorporate the data not used as final result however, we states the number of times we have done the experiments. If you want to read more about the different experiments you can read more here (link to wiki interlab page).

Week 26

Calibration 1: LUDOX – 27/06/18

OD600 was measured for the four replicates, and this was performed three times for the LUDOX and one time for ddH2O. The data from one of these replicants were used to the submission as a final result. Description and final results of the experiment can be found here (2.1 LUDOX).

Selma, Attila, Eirikur and Lasse

Calibration 2: Silica beads (1) – 27/06/18

OD600 was measured for the dilutions of silica beads. The results from this measurement was not used. Description and final results of the experiment can be found here (2.2 Silica beads).

Selma, Attila, Eirikur and Lasse

Transformation of E. coli DH5 α with the six test devices (1) – 27/06/18

Competent E. coli DH5 α were transformed with six test devices obtained from the distribution kit. The protocol used for the transformation were the one produced by iGEM, see here http://parts.igem.org/Help:Protocols/Transformation. Changes from the protocol were: - The use of LB media instead of soc in step 10. These transformants were not used.

Selma, Attila, Eirikur and Lasse

Transformation of E. coli DH5 α with the six test devices (2) – 28/06/18

Competent E. coli DH5 α were transformed with six test devices obtained from the distribution kit. The protocol used for the transformation were the one produced by iGEM, see here http://parts.igem.org/Help:Protocols/Transformation. Changes from the protocol were: - The use of LB media instead of soc in step 10. These transformants were used for the rest of the interlab experiments.

Selma, Attila, Eirikur and Lasse

Calibration 3: Fluorescein (1)– 01/07/18

The pH of PBS buffer from common stock was adjusted to pH=7,45.

The fluorescence was measured for the dilutions of fluorescein. The results from this measurement was not used. Description and final results of the experiment can be found here (2.3 Fluorescein).

Selma, Attila and Eirikur

Calibration 2: Silica beads (2) – 01/07/18

OD₆₀₀ was measured for the dilutions of silica beads. The results from this measurement was used for submission. Description and final results of the experiment can be found here (2.2 Silica beads).

Selma, Attila and Eirikur

Cell measurement: Overnight of transformants (1) – 01/07/18

An overnight of the transformants were set over in 5 mL LB + Chloramphenicol (CAM) (1:1000).

Selma, Attila and Eirikur

Week 27

Cell measurement: Cell growth, sampling, and assay (1) – 02/07/18

The overnight were diluted, abs₆₀₀ measured and diluted further to an abs₆₀₀ og 0,02 in a final volume of 12 mL. The samples were not measured and the experiment was therefore performed again. Description and final results of the experiment can be found here (3 Absorbance and fluorescence of transformed cells).

Selma, Attila, Eirikur and Lasse

Cell measurement: Overnight of transformants (2) – 02/07/18

An overnight was made from the overnight from the same day (Cell measurement: Overnight of transformants (1) – 01/07/18).

Selma, Attila, Eirikur and Lasse

Cell measurement: Cell growth, sampling, and assay (2) – 03/07/18

The overnight were diluted, abs₆₀₀ measured and diluted further to an abs₆₀₀ og 0,02 in a final volume of 12 mL. Abs₆₀₀ and fluorescence were measured. The results from this measurement was used for submission. Description and final results of the experiment can be found here (3 Absorbance and fluorescence of transformed cells).

Selma, Attila, Eirikur and Lasse

CFU per 0,1 OD₆₀₀: Starting sample preparation and dilution series (1) – 03/07/18

Abs₆₀₀ were measured on samples from the overnight culture (Overnight of transformants (2)). Triplicates were made after measurement of OD₆₀₀=0,1. After the dilution series the samples were plated. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD₆₀₀ E. coli cultures).

Selma, Attila, Eirikur and Lasse

CFU per 0,1 OD₆₀₀: CFU/mL/OD calculation (1) – 03/07/18

The plates were counted, but as we figured that the triplicate was not correct, we did not use the result. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD₆₀₀ E. coli cultures).

Selma, Attila, Eirikur and Lasse

CFU per 0,1 OD₆₀₀: Overnight culture (2) – 03/07/18

An overnight culture was made from the transformants.

Selma, Attila, Eirikur and Lasse

CFU per 0,1 OD₆₀₀: Starting sample preparation and dilution series (2) – 04/07/18

Abs₆₀₀ were measured on samples from the overnight culture. Triplicates were made and each of the triplicates were diluted to an OD₆₀₀=0,1. After the dilution series the samples were plated. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD₆₀₀ E. coli cultures).

Attila and Lasse

CFU per 0,1 OD₆₀₀: CFU/mL/OD calculation (2) – 05/07/18

The plates were counted, but as we were not satisfied with the result, we did not use the result and decided to do it once more. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD₆₀₀ E. coli cultures).

Attila and Lasse

Week 29

CFU per 0,1 OD₆₀₀: Overnight culture (3) – 16/07/18

An overnight culture was made from the transformants.

Attila and Lasse

CFU per 0,1 OD₆₀₀: Starting sample preparation and dilution series (3) – 17/07/18

Abs₆₀₀ were measured on samples from the overnight culture. Triplicates were made and each of the triplicates were diluted to an OD₆₀₀=0,1. After the dilution series the samples were plated. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD₆₀₀ E. coli cultures).

Attila and Lasse

CFU per 0,1 OD₆₀₀: CFU/mL/OD calculation (3) – 18/07/18

The plates were counted, but as we were not satisfied with the result, we did not use the result and decided to do it once more. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD₆₀₀ E. coli cultures).

Attila and Lasse

CFU per 0,1 OD₆₀₀: Overnight culture (4) – 18/07/18

An overnight culture was made from the transformants.

Attila and Lasse

CFU per 0,1 OD₆₀₀: Starting sample preparation and dilution series (4) – 19/07/18

Abs₆₀₀ were measured on samples from the overnight culture. Triplicates were made and each of the triplicates were diluted to an OD₆₀₀=0,1. The calculation for the OD₆₀₀=0,1 were made based on an average of the measurements of each of the singletons from the triplicate. After the dilution series the samples were plated. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD₆₀₀ E. coli cultures).

Attila and Lasse

CFU per 0,1 OD₆₀₀: CFU/mL/OD calculation (4) – 20/07/18

The plates were counted and The results from this measurement/calculation was used for submission. Description and final results of the experiment can be found here (4 Colony Forming Units per 0.1 OD₆₀₀ E. coli cultures).

Attila and Lasse

Week 30

Calibration 3: Fluorescein (2)– 24/07/18

As we figured out that a wrong setting on the plate reader had been used for Calibration 3: Fluorescein (1) we performed the experiment again.

The pH of PBS buffer from common stock was adjusted to pH=7,45.

The fluorescence was measured for the dilutions of fluorescein. The results from this measurement was used for submission. Description and final results of the experiment can be found here (2.3 Fluorescein).

Attila and Lasse

Egg membrane experiment

Up to week 35

Egg yolk membrane extractions

Various attempts to cut the egg yolk and obtain the intact membrane.
Experimenting with "fixation" of the membrane on various surfaces, integrity of membrane, etc...

Note: fresh eggs have stronger membrane

Learning the "technique" and manufacturing appropriate tools (bend wire) for handling delicate membrane

Eric & Natt

Week 34

Manufacturing the chamber and testing the setup – 24-26/08/18

The polystyrene plastic cup (PS) was found to be the most appropriate material for our setup.

Small holes were made at the bottom of the upper cup with heated nail
Membrane was placed across the holes, left to settle and fixed with wax

The next step was testing the leakiness of the setup. Cups were filled with water up to 2 cm and left for 24h. Upon any sign of leakiness the membrane was discarded.

Eric

Week 35

Manufacturing the chamber and testing the setup2 – 1-2/09/18

Upon further testing the simple "gluing" the membrane with universal glue was found to be superior in comparison to "waxing".

Same testing for leakines was performed with similar amount of water and therefore hydrostatic pressure.
It was found out that letting the membres dry even for 1h causes leakiness upon further testing for leakiness. All leaky membranes were discarded.
4 membranes that passed all the tests were stored in a plastic box with wet tissue to preserve the humidity and prevent drying of the membranes.

Week 36

Preparing the bacteria – 6/09/18

For preliminary experiments the following strains of SIEC bacteria were used:

(All had chaperones and mCherry as a reporter protein)

numbering	Genetic material	Description
1.	I+, S+, C+	Fully functional strain
2.	I-,S+,C+	Strain with disabled injectisome
3.	I+,S-,C+	Strain with injectosome but without secretion signal

Legend:

I = injectosome, (+) means functional injectosome, while (-) means the plasmid is lacking promoter in front DNA that in encoding injectisome.
S = secretion signal, a sequence of 20 amino acids which is believed to provide selectivity when it comes to secretion via injectosome.
C = chaperone. In this experiment all strains had chaperone. It is believed that they facilitate unfolding of the protein prior to injecting, however we tried to validate this in some other experiments.

Strains were picked from the araginoze plates and and overnight culture was grown in 10mL of LB media + Chloramphenicol.

Incubated overnight at 28°C since the other incubator was broken (for 37°C)

Incubation– 7/09/18

After measuring OD600 of overnight cultures the calculated amount of arabinose and IPTG was added to get the final concentration of 1% (w/w%) for arabinose and 1mM for IPTG. After incubating for 2h at 28°C (the 37°C incubator was still out of function) the bacteria solution was poured over membrane (without centrifugation) so that the upper chamber was filled approximately to the half (5mL of culture).

In lower chamber LB was poured (approx 3mL) for easier comparison of fluorescence.

Measurements

The samples were taken after 20 and 47 hours and compared with LB media serving as a blank.

Team:UCopenhagen/Notebook

Lab notebook

Cloning and Plasmid Construction

Week 25

Competent cell preparation 1 – 19/06/18

Competent cell preparation 2 – 20/06/18

Test for competency – 21/06/18

Week 28

Culture SIEC and SIECΔp1 E. coli – 11/07/18

Culture SIEC and SIECΔp1 E. coli –12/07/18

Prepare SIEC and SIECΔp1 competent cell stocks – 13/07/18

Week 29

pBAD and pTac Transformation – 16/07/18

PCR reporter genes and T3SS signal – 17/07/18

PCR reporter genes – 18/07/18

Restriction enzyme digestion – 19/07/18

Ligation and Cloning – 13/07/18

Week 28

Cloning biobricks – 19/09/18

Protoplast experiment with plants

Week 36

Further preparation for pilot experiment – 07/09/18

Week 37

Preparation of pilot experiment – 10/09/18

Pilot experiment – 11/09/18

Onion preparation– 25/09/18

Further preparation of onion sheets and tobacco plant observations– 27/09/18

Osmolarity test on onion sheets and tobacco plant – 02/10/18

Onion and tobacco assay – 05/10/18

Week 41

Remaking protoplast and preparing bacteria– 10/10/18

Remaking protoplast and preparing bacteria– 10/10/18

Inducing bacteria

Remaking onion and tobacco protoplasts at pH 5,0

Week 41

Washing protoplasts to remove bacteria – 11/10/18

Using Lyzozyme to remove bacteria – 11/10/18

Leakiness characterization

Week 36

Overnight of strains (1) – 06/09/18

Induction of strains + sample taking over time (1) – 07/09/18

Induction of strains + sample taking over time (1) – 08/09/18

Week 37

Overnight of strains (2) – 13/09/18

Induction of strains + sample taking over time (2) – 14/09/18

Induction of strains + sample taking over time (2) – 15/09/18

Induction of strains + sample taking over time (2) – 16/09/18

Induction of strains + sample taking over time (2) – 17/09/18

Induction of strains + sample taking over time (2) – 18/09/18

Induction of strains + sample taking over time (2) – 19/09/18

Induction of strains + sample taking over time (2) – 20/09/18

Measurement of fluorescence of supernatant and pellet (2) – 21/09/18

Experiments with supported lipid bilayers

Week 28

Learning to make liposomes – 12/07/18

Week 34

Transforming SIEC strains with GFP plasmids – 24/08/18

Week 35

Testing transformed bacteria for fluorescence – 27/08/18

Making lipid film stocks 1 – 29/08/18

Microscope tryout – 30/08/18

Bacteria incubation

Preparation of supported lipid bilayer

Microscopy

Making lipid film stocks 2 – 31/08/18

Week 36

Making lipid film stocks 3 – 05/09/18

Selma

Streaking bacteria – 05/09/18

Week 37

Full protocol tryout 1 – 12/09/18

InterLab

Week 26

Calibration 1: LUDOX – 27/06/18

Calibration 2: Silica beads (1) – 27/06/18

Transformation of E. coli DH5 α with the six test devices (1) – 27/06/18

Transformation of E. coli DH5 α with the six test devices (2) – 28/06/18

Calibration 3: Fluorescein (1)– 01/07/18

Calibration 2: Silica beads (2) – 01/07/18

Cell measurement: Overnight of transformants (1) – 01/07/18

CFU per 0,1 OD₆₀₀: Starting sample preparation and dilution series (1) – 03/07/18

CFU per 0,1 OD₆₀₀: CFU/mL/OD calculation (1) – 03/07/18

CFU per 0,1 OD₆₀₀: Overnight culture (2) – 03/07/18

CFU per 0,1 OD₆₀₀: Starting sample preparation and dilution series (2) – 04/07/18

CFU per 0,1 OD₆₀₀: CFU/mL/OD calculation (2) – 05/07/18

CFU per 0,1 OD₆₀₀: Overnight culture (3) – 16/07/18

CFU per 0,1 OD₆₀₀: Starting sample preparation and dilution series (3) – 17/07/18

CFU per 0,1 OD₆₀₀: CFU/mL/OD calculation (3) – 18/07/18

CFU per 0,1 OD₆₀₀: Overnight culture (4) – 18/07/18

CFU per 0,1 OD₆₀₀: Starting sample preparation and dilution series (4) – 19/07/18

CFU per 0,1 OD₆₀₀: CFU/mL/OD calculation (4) – 20/07/18