Team:Manchester/Wet-lab

Lab Protocols

View the detailed lab protocols we used when conducting experiments for UriGel

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Introduction Promoter Fluorescence Antibiofilm Mechanism Back to top

Introduction

To prevent the release of our engineered bacteria in the environment, we designed a genetic kill switch . The switch was designed to be dependent on two metabolites that are commonly found in urine: urea and sarcosine. A literature research revealed that a biosensor for urea had been previously designed by iGEM NYMU-Taipei 2008 ). However, no experimental data had been provided. To the best of our knowledge a biosensor for sarcosine has not been reported in literature. However, transcriptional regulators, being able to sense sarcosine, and the promoters that are controlled by these regulators, have been described. (Nishiya, Y. and Imanaka, 1996.)

The first step to construct our kill switch was to build biosensors for urea and sarcosine. The urea sensor was reconstructed using the information provided by iGEM NYMU-Taipei 2008. The sarcosine sensor was built from scratch.

Our objective was to characterise the urea and sarcosine sensors used for our kill switch design. To do this we have laid out plans to express red fluorescent protein (RFP) with the urea (BBa_K3710004) and sarcosine (BBa_K3710005) sensor we have designed. The results from these experiments can be found here: https://2021.igem.org/Team:Manchester/Wet-lab-results

We created a kill switch design which is described in more detail on our safety page. Due to the COVID-19 pandemic, we were unable to perform the wet lab experiments ourselves. However, our project advisors Dr Erik Hanko & Katherine Baker kindly agreed to perform them for us instead

References

https://www.protocols.io/view/igem-calibration-protocol-red-fluorescent-proteins-bcdjis4n?step=1&comment_id=86342
https://static.igem.org/mediawiki/2020/6/6e/T--Stockholm--Fluorescence.pdf
https://www.sciencedirect.com/science/article/pii/S0076687919300370
Nishiya, Y. and Imanaka, T. (1996). Analysis of a negative regulator, soxR, for the Arthrobacter sarcosine oxidase gene. Journal of fermentation and bioengineering, 81(1), pp.64–67.

Promoter Fluorescence Assay

PCR

Objective: Amplify synthesised urea and sarcosine biosensors by PCR

Initially assemble all reaction components on ice before transferring to pre heated thermal cycler for denaturation at 98 C

Component	25μL Reaction	50μL Reaction	Final Concentration
2X Q5 Master Mix	12.5 μL	25 μL	1X
10 uM Forward Primer	1.25 μL	2.5 μL	0.5 μM
10 μM Reverse Primer	1.25 μL	2.5 μL	0.5 μM
Template DNA (1ng/μL)	1 μL	2 μL	< 1,000 ng
Nuclease-free water	to 25 μL	to 50 μL	-

Thermal cycler conditions:

Step	Temperature	Time
Initial Denaturation	98^oC	30 Seconds
35 Cycles	98^oC / 50-72^oC / 72^oC	10s, 30s, 30s/kb
Final Extension	72^oC	2 minutes
Hold	10^oC	-

Run the PCR product on 1% w/v agarose gel
1. Mix 50 μL PCR product with 10 μL 6x loading dye (from NEB)
2. Load onto gel
3. Run the gel for 40 minutes at 120 V in 1xTAE buffer
Extract the PCR product from the gel using Qiagen gel extraction kit.
Quantify the DNA using Nanodrop

Cloning

Objective: Insert amplified urea and sarcosine sensors into a reporter vector harbouring the red fluorescent reporter gene.

Digest Bglbrick vectors pBbE8c-rfp (colE1 ori) and pBbS8c-rfp and PCR product with AatII and Ndel over night at 37 C.

Component	Volume
1 ug insert DNA	*Depends on DNA extract concentration
1 ug vector DNA	*Depends on DNA extract concentration
10x CutSmart Buffer	5 μL
Ndel	1 μL
AatII	1 μL
Water	Make up to final volume of 50 μL

Run the digested insert DNA and vector on an agarose gel following the instruction above. Excise the bands of the correct size and extract the DNA using the Qiagen gel extraction kit.
Set up ligation reaction mixture in microfuge tube on ice according to the manufacturer's protocol

Component	20 μL Reaction
Quick Ligase Reaction Buffer (2X)	10 μL
Vector DNA	50 ng
Insert DNA	37.5 ng
Nuclease-free water	to 20 μL
Quick ligase	1 μL

Gently mix the reaction components with a pipette
Incubate at room temperature for 2 hours.

Transform 4 μL ligated product into 50 μL E. coli DH5-alpha using heat shock method. Following manufacturer instructions: https://international.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c2987
Spread 50 μL transformed strains on LB agar plates containing 100 ug/mL ampicilin and incubate 37 C for 18 hours.
Test for correctly transformed colonies by growing the transformed strains on plates containing the following supplements:

Plate	Expected colour if cloning was successful
LB agar	Off-white (Depending on how leaky the promoter is)
LB Agar + Arabinose 0.1% w/w	Same as control above
LB Agar + Inducer(High Concentration)	White if promoter is not responding to the inducer. Red/pink cells (if the promoter is responding to the inducer)

Fluorescence Reporter Assay

Objective: Quantify the promoter activity of the urea and sarcosine sensors in response to different concentrations of their inducers.

Protocol:

A single colony of E. coli DH5a, harboring the urea or sarcosine biosensors, was used to inoculate 5 mL of M9 minimal medium (containing the following per liter: 200 mL of 5 × M9 salts [64 g/L Na2HPO4·7H2O, 15 g/L KH2PO4, 2.5 g/L NaCl, 5 g/L NH4Cl], 2 mL of 1 M MgSO4, 0.1 mL of 1 M CaCl2) supplemented with 1 μg/mL thiamine, 20 μg/mL uracil (Jensen, 1993), 25 μg/mL chloramphenicol, 0.2% casamino acids and 0.8% (w/v) glucose.
Grow cells for 18 h in a 50-mL conical centrifuge tube with orbital shaking at 37 °C and 200 rpm
Dilute the preculture 1:40 into 5 mL of fresh medium and incubate at 37 °C and 200 rpm
At an OD600 of 0.2, transfer 142.5 μL of cell culture into a 96-well plate (black with clear bottom)
Add 7.5 μL of stock inducer at the desired concentration. For example, add 7.5 μL of 100 mM urea to achieve a final concentration of 5 mM in the cell culture
Using a plate reader, measure the RFP fluorescence using 585 nm as excitation wavelength and 620 nm as emission wavelength both with a 10 nm bandwidth. Set the gain to 100% unless the fluorescence reaches the detection limit. Set the flash frequency to 100 Hz and the integration and lag time to 20 μs and 0 μs, respectively. At the same time, measure the absorbance using a wavelength of 600 nm with a 5 nm bandwidth. For both fluorescence and absorbance measurements, set additional acquisition parameters to: number of flashes of 10 and settle time of 10 ms. The temperature of the plate reader set to 37 °C with orbital shaking at a frequency of 582 rpm and an amplitude of 1 mm. Take measurements at 0, 1h, 2h, 4h, 6h, 8h, 24h

Antibiofilm Mechanism Characterisation

PCR

We were unable to test the action of our dispersin B during the project due to the Covid-19 pandemic, but we planned protocols for the experiments to do so:

Initially assemble all reaction components on ice before transferring to pre heated thermal cycler for denaturation at 98 C

Component	25μL Reaction	50μL Reaction	Final Concentration
2X Q5 Master Mix	12.5 μL	25 μL	1X
10 uM Forward Primer	1.25 μL	2.5 μL	0.5 uM
10 uM Reverse Primer	1.25 μL	2.5 μL	0.5 uM
Template DNA (1ng/μL)	1 μL	2 μL	< 1,000 ng
Nuclease-free water	to 25 μL	to 50 μL	-

Thermal cycler conditions:

Step	Temperature	Time
Initial Denaturation	98 C	30 Seconds
35 Cycles	98 C / 50-72 C / 72C	10s / 30s / 30s/kb
Final Extension	>72 C	2 minutes
Hold	10 C	-

Run the PCR product on 1& w/v agarose gel
1. Mix 50 μL PCR product with 10 μL 6x loading dye (from NEB)
2. Load onto gel
3. Run the gel for 40 minutes at 120 V in 1xTEA buffer
Extract the PCR product from the gel using Quiagen gel extraction kit: https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/dna-clean-up/qiaquick-gel-extraction-kit/?catno=28704
Quantify the DNA using nanodrop

Cloning

Digest BglBiobrick vectors pBbE8c-rfp (colE1 ori) and pBbS8c-rfp and PCR product with AatII and Ndel over night at 37 C

Species	Volume
1 ug insert DNA	*Depends on DNA extract concentration
1 ug vector DNA	*Depends on DNA extract concentration
10x CutSmart Buffer	5 μL
Ndel	1 μL
AatII	1 μL
Water	Make up to final volume of 50 μL

Purify the insert DNA and vector using agarose gel electrophoresis (1% w/v). Extract the bands of the correct size using the Qiagen gel extraction kit.
Set up ligation reaction mixture in microfuge tube on ice https://international.neb.com/protocols/0001/01/01/quick-ligation-protocol:

Component	20 μL Reaction
Quick Ligase Reaction Buffer (2X)	10 μL
Vector DNA	50 ng
Insert DNA	37.5 ng
Nuclease-free water	to 20 μL
Quick ligase	1 μL

Gently mix the reaction components with a pipette
Incubate at room temperature for 5 minutes
Incubate at room temperature for 5 minutes

Transform 1-5 μL ligated product into 50 μL E. coli DH5-alpha using heat shock method. Following manufacturer instructions: https://international.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c2987
Spread 50 μL transformed strains on LB agar plates containing 100 ug/mL ampicilin and incubate 37 C for 18 hours.
Test for transformed colonies by growing on agar plate with antibiotic corresponding to the chosen antibiotic resistance gene.

Coculture Biofilm Assay

Reference: https://doi.org/10.1002/9780471729259.mc01b01s22

Coculture a transformed E. coli DH5-alpha colony (Lsr-DspB) with a second non-transformed E coli strain (MG1566) in 5mL culture medium.
- Add various concentrations of each strain:
- 0, 1000, 10,000, 100,000 CFU/ml of the transformed DH5-alpha strain with 0, 1000, 10,000 and 100,000 non-transformed E. coli stain MG1566. This will be 16 different samples, so with 3 replicates that is 48 samples total.
Dilute cultures 1:100. Add 100 μL of each diluted culture into microtiter plate
Cover the plate and incubate at 25C for 72 hours
Inoculate biofilm assay plates directly in 100 μL culture medium from the overnight microtitre plates and incubate at 25C overnight.
Set up four small trays and add 1-2 inches of tap water to the last three
Remove planktonic bacteria from each microtiter dish by briskly shaking the dish out over the waste tray. To wash wells, submerge the plate in the first water tray and then vigorously shake out the liquid over the waste tray. Replace water when it becomes cloudy
Add 125 μL of 0.1% crystal violet solution to each well. Stain 10 min at room temperature.
Shake each microtiter dish over the waste tray Wash the dishes in the next two water trays and shake out as much liquid as possible after each wash
Invert each microtiter dish and vigorously tap on paper towel to remove any excess liquid
Allow plates to air dry
Add 200 μL of 30% acetic acid to each stained well. Allow the dye to solubilise by covering the plates and incubating 10-15 minutes at room temperature
Briefly mix the contents of each well with a pipette then transfer 125 μL of the acetic acid/crystal violet solution from each well into a separate well in an optically clear flat bottom 96-well plate. Measure optical density from 500nm to 600nm for each sample. Be sure to add a zero-well with no biofilm and only acetic acid/crystal violet mix.