Experiments with biomimetic membranes

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.

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.

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.

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.

*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).

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.

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.

*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:

**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.

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

Vials were kept in -20°C.

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.

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.

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.

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).

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).

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.

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.

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).

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).

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).

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).

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).

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).

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).

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).

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

An overnight culture was made from the transformants.

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).

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).

Week 29

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

An overnight culture was made from the transformants.

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).

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).

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

An overnight culture was made from the transformants.

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).

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).

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).

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

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.

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)

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!)