Experiments

LAB SAFETY

Laboratory safety was a primary focus in all the experiments we conducted. During the design of the project, considerable time was spent in ensuring we do not have to rely on pathogenic strains for any of the experiments, especially in our final engineered bacteria which we propose to employ in a bioreactor. We made sure our team and project comply with the guidelines set by iGEM and IISc’s Office of Laboratory Safety and Environmental Health (OLSEH). Our experimental designs passed through thorough checks of our PI and safety advisors. Throughout the project, our PhD advisors and other lab members supported us whenever we had a concern about safety.

Biology Lab Safety

All the members of the wet lab team underwent rigorous training sessions for a week with the undergraduate lab instructors. Minute details about sterilization and containment were noted. The instructors also advised us on key safety aspects throughout the project.
Gloves, lab coat, full-length trousers and closed-toe shoes were made mandatory to work in the lab.
Food and drinks were strictly prohibited.
All activities with bacterial cultures were performed under a laminar hood. The hood was frequently sterilized with 30 minutes of UV.
The entire lab including the hoods and the work-benches were thoroughly cleaned every Monday morning.

Chemistry Lab Safety

Working with Halocarbons - Halocarbons must be used safely under the chemical hood and gloves, splash goggles and lab coat must be worn at all times. Some of them like TCE are known carcinogens and its exposure should be completely avoided.
Gloves were always used during the experiment and all experiments were done under a chemical fume hood with laminar air flow. To kill off bacteria and wash the working station, 70% ethanol solution was used and bleach along with autoclave was used to sanitise the equipment.
Diethyl ether is extremely volatile and flammable and must be kept away from any ignition or electricity source. It should be stored in the dark as it can produce explosive peroxides when exposed to light and air.
Safety while using GC - It is very important to prevent the entry of Bacteria and very polar material into the GC, thereby a non-polar organic solvent like diethyl ether is used to phase out the halocarbons. GC must be carefully switched on and carefully cooled down. The maximum concentration of a substance in the GC is 0.7mg/mL and this was an important consideration for all our calculations.

Microorganisms and Parts

All the members of the wet lab team underwent rigorous training sessions for a week with the undergraduate lab instructors. Minute details about sterilization and containment were noted. The instructors also advised us on key safety aspects throughout the project.
Gloves, lab coat, full-length trousers and closed-toe shoes were made mandatory to work in the lab.
Food and drinks were strictly prohibited.
All activities with bacterial cultures were performed under a laminar hood. The hood was frequently sterilized with 30 minutes of UV.
The entire lab including the hoods and the work-benches were thoroughly cleaned every Monday morning.

BACTERIAL CULTURES AND STOCKS

Inoculation:

For the very first inoculation, we scraped the top of the slant with a sterile loop and then, without touching the walls of the test tube, we made sure to dissolve the colony on the loop in the LB that was in the test tube (5 ml of LB was used)
For rest of the inoculations, we picked a single colony out of the streak plate that we wanted to inoculate, and inoculated it in a test tube containing LB (amount of it depends on what we want to do with the inoculations afterwards)

Streak plating:

Depending on the purpose, we took a pipette with pipette tips and we either scraped the top of glycerol stock or we dipped the tip in the inoculation
We then carefully streak some lines in one quadrant and then discard the tip
With a fresh tip, we start streaking the second set of lines with some of the first lines as the start point and streak into the second quadrant
We repeat this process for about 4 times making sure that the lines don’t overlap to make a good streak plate

Spread plating:

Take a bit of the culture that is to be plated, and put it in the center of the plate
We spread it uniformly using a sterile spreader until there is no excess culture present on the plate

Glycerol stock preparation:

In a 2ml tube, we take 500 micro-litres of 50% glycerol and 500 micro-litres of overnight culture.
We then keep these tubes in a labelled box and store it at -80^o C.

Chemically competent cell preparation:

On the night prior to competent cell preparation, we inoculate for an overnight culture in LB
We use 1 ml of the overnight culture to prepare a secondary culture in 100ml LB in a 500 ml flask
We incubate the secondary culture in a 370 incubator until the OD reaches 0.4-0.6
We chill the secondary culture on ice for 15 minutes, and then split the secondary culture to 2*50 ml Falcon Tubes
The cells will continue to be kept in ice from now on
We centrifuge the cells at 4^oC for 15 minutes at 4500 rpm, then decant the supernatant and resuspend each tube in 10 ml of 0.1M ice cold CaCl2
We then incubate this mixture in ice for 20 minutes and again centrifuge at 4^oC for 15 minutes at 4500 rpm
We decant the supernatant, resuspend in 10 ml of ice cold 0.1 M CaCl2 and incubate on ice for 30 minutes
We then centrifuge it again at 4^oC for 5 minutes at 4500 rpm
We now decant the supernatant and resuspend each tube in 2ml of 0.1M CaCl2/15% glycerol solution and then aliquot 100 micro-litres of these cells in micro centrifuge tubes and then flash freeze them using liquid N2
Store the flash frozen cells in -800 C

Transformation:

Place the competent cells (100 micro-litre) aliquots on ice and allow them to thaw
Add 1 – 5 microlites of the DNA to be transformed to the thawed competent cells and then mix it well by tapping the tube
We then keep this mixture on ice for t 30 minutes and when done, we give it a heat shock of 42^o C for 60-90 s and immediately keep it on ice
Let the mixture stay on ice for 15 minutes, and then add 1 ml of LB media for recovery
Keep the above mixture in a rotatory incubator and incubate for 1 hour
We then centrifuge it at 4500 rpm for 3 minutes and 30 seconds, decant ~900 micro-litres of the supernatant and resuspend the pellet in the remaining supernatant
Plate this resuspension on an appropriate plate using the spread plating method.

Plasmid isolation:

We inoculate the bacteria that we want to isolate plasmids from, on the night prior
We take the overnight culture and transfer it to multiple Micro Centrifuge Tubes (MCTs)
We centrifuge these MCTs at 13,000 rpm for 3 minutes and then completely discard the supernatant
We take P1 buffer (as present in the kit) and add 250 micro-litres of it to one of the tubes, resuspend, then transfer all the contents to another tube, transfer….and we repeat this until all the cell pellets are resuspended in the 250 micro-litres of P1 buffer
We then add P2 buffer to the MCT, shake it and allow it for at least 2 minutes and at most 5 minutes
We then add N3 buffer, shake the MCT well and then centrifuge this for 10 minutes at 13,000 rpm.
We then take the supernatant out and add it to the DNA column, and centrifuge it again at 13,000 rpm for 1 minute.
We then add buffer PE to the column and centrifuge it at 13,000 rpm for 1 minute
We then dry spin it again for 90 seconds to remove excess buffer and then place the DNA column into a new MCT
We add 20-25 micro-litres of preheated (to 500 C) Elution buffer to the DNA column, wait for 10 minutes and then spin it down for 1 minute at 13,000 rpm.

PCR AND CLONING

PCR:

Choose the appropriate enzyme that suits our purpose of performing the PCR and the primers designed
Create a mastermix for PCR that contains the dNTP mix, Forward Primer, Reverse Primer, the respective Polymerase enzyme and Autoclaved Milli-Q water, and then we add the template to the mix
Set the temperature and time for denaturing, annealing and extension respectively appropriately and leave it in the PCR machine for the required amount of time
Run the reaction on gel and verify the accuracy of the PCR reaction

Fusion PCR:

First, the insert is PCR-amplified with the chimeric primers so that the final PCR product has overlapping regions with the vector.
Then, vector and insert are mixed, denatured and annealed; the hybridized insert then is extended by polymerase using vector as a template until polymerase reaches 5′ end of the insert. After several PCR cycles, the new plasmid with two nicks (one on each strand) gets accumulated as a product.
The new plasmid can be transformed into after the parental plasmid is destroyed by a single digest.

Colony PCR:

First suspend the colony that we want to perform colony PCR on, in 10 microlitres of Autoclaved Milli-Q water
Repeat the steps done for PCR but be aware of the temperatures and times

Gibson Cloning:

If we are setting up an ‘X’ micro-litres Gibson reaction, take X/2 micro-litres of Gibson mix and put it in a PCR tube.
We also fill make sure that the vector:insert ratio is at least 1:2 and ideally 1:3, and the combined volume of insert and vector should be X/2
We take the PCR tube containing the Master mix, vector and insert, and keep it to incubate at 50^oC for 1 hour
We take the PCR tube out of the incubator and transform it to the desired comp cells and check for successful colonies

Restriction Digestion:

Make a mastermix which contains the Restriction Enzyme, the apt buffer for that enzyme and Autoclaved Milli-Q water (the amount of enzyme depends on the amount of vector)
Add the vector to be digested to the mastermix and leave it for an appropriate amount of time (depends on the length of the vector) at the optimum temperature of the enzyme.

Gel Extraction:

Excise the gel fragment with a scalpel, transfer it to an MCT and add 0.6 ml of buffer QG to it (we used Qiagen gel extraction kit for all the steps)
Incubate at 500C for about 5-6 minutes and we invert the tubes every 2 minutes
After the gel is completely dissolved in buffer QG, we add 0.3 ml of isopropanol to the MCT with the above mixture, and invert it 10-15 times
Place the spin column on collection tube and add 0.7ml of the sample at a time and centrifuge it for 40 seconds at 14,000 rpm
Discard the flowthrough and add the rest of the sample to the spin column and centrifuge again for 40s at 14,000 rpm
We now add 0.75 ml of buffer PE to the spin column and centrifuge for 1 minute at 14,000 rpm
Discard the flowthrough and dry spin it again for 1 minute at 14,000 rpm
We then place the column in an MCT and add 15-25 micro-litres of preheated Elution buffer to the center of the column
We wait for 10-15 minutes and then centrifuge again for 1 minute at 14,000 rpm

Ligation Reaction:

We take the fragments to be ligated and make sure that they have proper sites that ensure efficient ligation
We then create a mastermix with the appropriate amounts of Ligase buffer, Autoclaved Milli-Q water and the ligase enzyme
We then mix the fragments to be ligated in the ligation master mix and leave it at 16^oC for 16 hours

Annealed Oligo Cloning:

First, the vector is double-digested with restriction enzymes and stored. A primer set is designed such that, when annealed, the forward and reverse primers produce a dsDNA fragment with the sequence to be inserted and ssDNA overhangs on either end that are compatible with the sticky ends on the digested vector.
The primers are annealed by resuspending in STE buffer, heating to 95^oC for 3-5 minutes, then letting it cool to room temperature over ~45 minutes. If multiple oligo fragments are being assembled together, the forward primers must be 5’ phosphorylated using T4 polynucleotide kinase before annealing.
The annealed oligos and the digested vector are ligated overnight at a 3:1 ratio of insert:vector by mass, then used to transform competent cells.

PROMOTER ACTIVITY ASSAYS

We developed a protocol to measure the activity of promoters under hypoxic conditions by quantifying sfGFP expression with a fluorescence microscope. The strong constitutive promoter EM7 was used as a reference to control for the effect of hypoxia on cells.

Recombinant bacteria carrying a plasmid with the promoter of interest cloned upstream of the sfGFP reporter sequence are generated. These are grown overnight under aerobic conditions in LB media at 37oC and 180 RPM with the appropriate antibiotic. The stationary-phase culture is diluted to an O.D. of 0.80 in LB media fortified with 4mM cysteine to promote anaerobic growth.
Hypoxic conditions are created in a glass tube as specified in the chemical assays section. Using a needle syringe, 2mL of this diluted culture is added to both the hypoxic glass tube as well as an identical aerobic glass tube that is capped, but not sealed. The hypoxic and aerobic cultures are incubated in the shaker-incubator at 37^oC and 120 RPM for six hours.
1mL of culture from each tube is taken into a microfuge tube and centrifuged at 3000 RPM for 2:00 minutes. 900uL of the supernatant is removed and the pellet is resuspended in the remaining volume. 2uL of this is spotted onto a clean glass slide and covered with a cover slip.
The slides are imaged with a fluorescence microscope at 100X or greater with identical exposure times and emission intensities. Images are quantified using FIJI to measure the change in fluorescence intensity in individual bacterial cells between the aerobic culture and the hypoxic culture for each promoter, which can be used to estimate promoter activity.

BIOFILM ASSAY

Add 3 ml of LB to each well of all the seven 12-well plates.
For temperature and Antibiotic Stress,

Take five plates from the previous step.
To the second column, add Amp (concentration, as indicated in diagram) and third column, add Strep (concentration, as indicated in diagram).
Now, to the first three columns, add 30 uL of bacterial inoculum, leave the last column empty for control.
Place these five plates in different temperatures for incubation for 48 hours, as listed (15°C, 20°C, 25°C, 30°C, 35°C)

For variable Antibiotic stress,

Take two 12-well plates from the initial step.
Prepare serial dilutions of the antibiotics (one plate for amp, another for strep) in a 2x manner and add to the wells as indicated in the diagram.
To the second and third row, add 30 uL of bacterial inoculum for replicates and leave the first row as control.
Put at 30°C for incubation for 48 hours.

After this, throw the methanol out and add 3 ml of 0.1 or 1% crystal violet stain and incubate for 20 minutes at room temperature.
Rinse the plate with 1x PBS twice.
Add 3 ml of 70% ethanol to each well and measure the OD at 570 nm.

CHEMICAL ASSAYS

Herein we outline the protocol developed and optimized to study the rate of degradation of any halocarbon (in liquid or stock solution form) using GC (Gas Chromatography). We use TCE (trichloroethylene) as a model substrate.

The first step involves creation of hypoxic conditions. This involves connecting a round-bottom flask with a magnetic stirrer to a two-way adapter and then connecting that to a Schlenk line and an Argon balloon. The flask would be then flushed with argon and vacuum repeatedly several times to drive out all the air and put in argon. Argon is chosen in order to create anaerobic conditions since its heavier than air and if any air remains, it would be at the top.
Under argon, the two-way adapter is replaced with a Teflon septum (Teflon septum is used specifically since halocarbons dissolve through rubber, a tip given to us by Dr. Lawrence Wackett) with an argon balloon through it.
2mL of bacterial culture is transferred to this via a needle syringe. The OD of the culture is later standardized to 0.7 (to study the log phase) and 2.0 (to study the stationary phase). Cultures are grown on a standard LB broth.
Using a micropipette or a Hamilton syringe, 0.02mL (20 μL) of our halocarbon TCE (or corresponding amount of stock solution in case of gaseous halocarbons) is added to this and the stirrer is set up at a temp of 25 degrees C and 360rpm.
After desired time, corresponding amount of air is added to make the conditions aerobic. (We assume air has 20% oxygen so corresponding amount of oxygen is added)
At the end of every time stamp (we considered 0min, 20min, 40min and 60min timestamps), 10mL of an organic solvent is added and the flask is shaken to separate the organic and aqueous layers. (Choice of solvent is such that its non-polar enough to phase out from the aqueous medium and has a low boiling point so that there is good separation between the peaks of the solvent and our substrate (TCE), which on optimisation was found to be diethyl ether). The role of the non-polar solvent is to prevent the entry of any polar substance or cells into the GC column since polar substances tend to spoil the column as well as the readings
1ml of the supernatant is carefully abstracted using a 1mL syringe from the top layer as the organic solvent used is less dense than LB layer. This is then transferred to a 2mL glass vial used in the GC along with a known quantity of internal standard if concentration determination is required. (Around 2 μL of decane can be added as the internal standard after optimisation experiments).
Corresponding setting are set up in the gas chromatogram and the sample is run. Samples of various time stamps are collected, and we can obtain our (relative) concentration vs time curve by comparing the intensity or the area under the peak corresponding to the substrate (TCE) with time.
The time scale for the assays was decided as 1hr for TCE after looking at some reports of microbial TCE degradation rates.

Protocol and settings for GC-MS:

Once 1mL of sample with substrate (TCE) dissolved in the non-polar low boiling solvent (Diethyl Ether) is obtained in a glass vial, it is run in the GC apparatus with the following settings:

(Apparatus used – Agilent 8890 GC, Column HP-5)

Max oven temperature is set to 350 °C and Front SS Inlet N2 Heater is set to 250 °C.
After optimisation of settings to give maximum peak separation between the solvent Diethyl Ether, substrate TCE and internal standard Decane, we used the method: Initial oven temperature = 30 °C, Rate of temperature increase = 25 °C/min, Hold time = 2 minutes, Injection volume of 0.5 μL is used for each trial.
After each run, the chromatogram obtained is studied, and the expected peaks are marked. Further, an estimate of the amount of TCE in the sample is made by integrating the TCE peak.

Expected retention times when using the above-mentioned settings: TCE = 1.77 minutes, Decane = 4.7 minutes

Protocol and testing TCE toxicity on bacteria:

As TCE was reported to be toxic to the bacterial strain KT2440 at high concentrations, we decided to study the extent of toxicity. The protocol followed was:

In 4 round-bottom flasks, a magnetic bead was placed and using a syringe, 2mL of bacterial culture at OD = 0.7 (log phase) was transferred into each.
In the RBs, using a micropipette, different amounts of TCE in volume were added: 0mL, 0.05mL, 0.1mL and 0.2mL. Teflon septa were used to seal the flasks to prevent evaporation of TCE.
The RBs were allowed to stir for around 4-5 hours at room temperature. Then, bacterial samples from the bottom of the RB were collected for plating.
The relative number of colonies observed with varying TCE volumes were recorded and thus the toxicity of TCE on our strain could be estimated.