Lab notebook

The notebook is a chronological overview of what has happened in the lab. It is organized after each experiment.

Cloning

Making BioBricks

Liposome characterization

Onion characterization

Leakiness characterization

Membranes and microscope

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

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.

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.

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:

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

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.

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 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.

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.

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.

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.

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.

*This number is higher than 29/08/18. This means that the concentration for DOPC is not exact. Stock 2s were made:

**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.

Mixtures were aliquoted into glass vials to contain 0,2 μmol lipids:

Vials kept in -20oC.

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.

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

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.

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.

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).

Egg membrane experiment