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 Preparing liposomes protocol. We made some tryout liposomes, that were flashed freezed and stored in -20 oC.

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 with constitutive expression. 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_J36400
SIEC x BBa_I20270
SIECΔp1 x BBa_J36400
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 incubator before being kept in fridge. Conclusion: hereafter we used the bacteria transformed with BBa_I20270.

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 (not necessary). Incubated at 37 oC 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. RESULTS, distribution AND SETTINGS Glycerol stocks were made. Conclusion: because a negative control was forgotten, results were inconclusive.

Selma

Co-transformations of SIEC strains 1 – 28/08/18

We made co-transformed bacteria for the membrane experiments that needed bacteria with both a reporter to visualize the bacteria and and the T3SS signal tagged reporter. We used constitutive active GFP, BBa_I20260, in a plasmid with a kanamycin resistance gene found in the distribution kit plate 4 well 18A. For our signal tagged reporter, we used mCherry plasmids made the xx/xx/18 by Nat. Transformation as described in Transformation protocol until step 14, the following combinations of strain and plasmid was made:

T1: SIEC x plasmid plate 4: 18:A x ss-mCherry (repeats a and b)
T2: SIECΔp1 x plasmid plate 4: 18:A x ss-mCherry (repeats a and b)
T3: SIEC x plasmid plate 4: 18:A x Chaperone-ss-mCherry
T4: SIECΔp1 x plasmid plate 4: 18:A x Chaperone-ss-mCherry
T5: Mach1 x plasmid plate 4: 18:A

For the co-transformations plates were made with LB and agar with both kanamycin (50 μg/mL) (KAN) and CAM. Before plating, the bacteria was spinned down, 800 μL supernatant was discarded and bacteria resuspended. Mach1 with plasmid plate 4: 18:A were plated on CAM plates for backup of the plasmid. Plates with bacteria spent 15 hours in incubator. Colony count:

Transformation	Colonies
T1,a	3
T1,b	0
T2,a	1
T2,b	4
T3	0
T4	0

Conclusion: only co-transformations with plasmids without chaperones gave colonies.

Selma

Testing transformed bacteria for fluorescence – 29/08/18

To test if the transformation was successful fluorescence were measured on a plate reader. Colonies from transformation 28/08/18 was picked into 50 mL falcon tubes with 5 mL LB media that also contained 1% arabinose. Incubated at 37 oC at 130 rpm for 5 hours. Triplicates of each 100 μL of culture was pipetted into wells of a 96 well-plate. Fluorescence and absorbance was measured. RESULTS, SAMPLE DISTRIBUTION AND SETTINGS Conclusion: because a negative control was forgotten, results were inconclusive.

Selma and Nat

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 in this mass from producer. Lipids were dissolved in 1 mL chloroform. To store the lipids, we used nitrogen flow to exchange air in the glass vials with nitrogen. Because some of the solvent evaporated with the nitrogen flow, this was redone 31/08/18.

Second, we made biotins stock 2. We got biotin in a stock concentration of 0,5 μg/μL (stock 1). 150 μL of biotin stock 1 was added to 600 μL chloroform to make biotin stock 2.

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 confocal microscope in Nikos Hatzakis lab. Introduction 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). We added 10 μL of overday culture from the 29/08/18 to 5 mL LB media with CAM and KAN in 50 mL falcon tubes (SIEC and SIECΔp1 with GFP, BBa_I20270, and T3SS signal tagged mCherry) Put in incubator at 37oC 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 tryout, we used the liposomes made 12/07/18 (wrong membrane composition). Microscope slides were washed with tris buffer. Membrane contained dye atto655.

Microscopy:

Microscope slides was washed with the same media used for 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. PICTURESbacfrom microscope PC (SIEC with BBa_I20270) PICTURESmembranefrom microscope PC Membrane was not intact, probably because the slide was dropped on the floor.

Selma, Attila, Lasse and Nat

Making lipid film stocks 2 – 31/08/18

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 1s.

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. Stock 2s were made:

Lipid	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. From here 3 mixtures were made.

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	31 μL	28 μL

Vials kept in -20oC.

Selma

Streaking bacteria 1 – 02/09/18

Bacteria from 30/08/18 cultures (co-transformations 28/08/18) was streaked on plates with CAM and KAN. Incubated overnight and kept in fridge.

Attila

Co-transformations of SIEC strains 2 – 02/09/18

Co-transformations of with the plasmids containing chaperones was redone, see 28/08/18. Again transformation was done according to Transformation protocol until step 14, the following combinations of strain and plasmid was made: T3: SIEC x plasmid plate 4: 18:A x Chaperone-ss-mCherry T4: SIECΔp1 x plasmid plate 4: 18:A x Chaperone-ss-mCherry

Nat

Week 36

Making lipid film stocks 3 – 05/09/18

To finish lipid film stocks solvent was evaporated with nitrogen flow for about 5 minutes. Then vials were centrifuged on a spin-vac for 15 minutes. Vials kept in -20 oC.

Selma

Streaking bacteria 2 – 05/09/18

Bacteria from glycerol stock 27/08/18 (transformations 24/08/18) was streaked on plates with CAM and KAN. Incubated overnight and kept in fridge.

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.

(Don't ask about results!)

Team:UCopenhagen/Notebook

Lab notebook

Membranes and microscope

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

Co-transformations of SIEC strains 1 – 28/08/18

Testing transformed bacteria for fluorescence – 29/08/18

Making lipid film stocks 1 – 29/08/18

Microscope tryout – 30/08/18

Preparation of supported lipid bilayer:

Making lipid film stocks 2 – 31/08/18

Streaking bacteria 1 – 02/09/18

Co-transformations of SIEC strains 2 – 02/09/18

Week 36

Making lipid film stocks 3 – 05/09/18

Streaking bacteria 2 – 05/09/18

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

Week 27

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

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

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

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

Week 29

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

Week 30

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

Egg membrane experiment

Up to week 35

Egg yolk membrane extractions

Week 34

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

Week 35

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

Week 36

Preparing the bacteria – 6/09/18

Incubation– 7/09/18

Measurements

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