Experimentation and Documentation

iGEM Protocol Handbook

Click <a href="https://static.igem.org/mediawiki/2018/c/ce/T--Virginia--2018_Virginia_IGEM_Protocol_Handbook.pdf" target="_blank">here</a> to view a curated list of protocols Virginia iGEM has used and refined over the past few years.

Workflow

The general workflow of our team occurred as follows.

1) Creation of and testing of competent cells.
2) Part assembly using Golden Gate or Gibson assembly.
3) Transformation.
4) Colony picking for overnight cultures and restreaking.
5) Culture miniprep and glycerol stock creation.
6) Digestion and gel electrophoresis of miniprep product.
7) Growth and sampling for Flow Cytometry.
8) Creation of BioBrick.
9) Sequencing of final part.

Protocols

Chemically Competent E. Coli Cells

Materials:

• Pre-culture of XL1-blue, DH5α, or SCS
• 500 mL LB
• 2800 mL Fernbach flask
• 2.5 mL 2M MgCl2
• Cooling culture shaker
• Two 500 mL centrifuge bottle, autoclaved and chilled at 4C
• Liquid N2
• 80-160 1.5mL Eppendorf Tubes, autoclaved and chilled at 4C
• Cold Room
• 3 mL DMSO
• 500mL Transformation Buffer

Transformation Buffer:

1) Add 350 mL of MilliQ water.
2) Then add 15 mM CaCl2●2H2O (7.5mL of 1M).
3) Then add 250 mM KCl (50 mL of 2.5M).
4) Then add 10 mM PIPES-disodium salt pH 7.0 - adjust with KOH (10 mL of 0.5M).
5) Adjust pH to 7.0 with 1M KOH or NaOH.
6) Then add 55 mM MnCl2●4H2O (55 mL of 0.5M).
7) Fill up volume to 500mL using MililQ Water.
8) Filter Sterilize.
9) Chill at 4C before use.

Procedure:

1) Prepare an O/N culture of cells in 1 mL of LB at 37C incubating shaker.
2) Inoculate the O/N culture in 500 mL of LB in the 2800 mL Fernbach Flask.
3) Add 2.5 mL of 2M MgCl2 to the LB in the Fernbach flask.
4) Grow culture at 18C in the cooling shaker. Grow cells until the OD=0.12.
5) Take frequent measurements. Usually takes 30-40 hours for DH5a.
6) Recommended that you plot a curve to predict when the cells will be fully grown.
7) Transfer culture to centrifuge bottles and incubate on ice for 10 minutes.
8) Centrifuge at 5,000 rpm [SLA-1000 Rotor] for 10 minutes at -4C.
9) Decant supernatant and resuspend pellet in 150 mL transformation buffer.
10) Centrifuge at 5,000 rpm [SLA-1000 Rotor] for 10 minutes at -4C.
11) Decant supernatant and resuspend by gently pipetting the pellet up and down in 40 mL transformation buffer.
12) Add 3 mL DMSO. While adding the DMSO, swirl the solution of cells to prevent excess exposure to the DMSO.
13) Aliquot ~250-500 μL (recommend 300 μL) of cells to each Eppendorf tube. (500 μL will be more than what is needed for most transformations, but will require less pipetting, tubes, and freezer boxes.).
14) After each Eppendorf tube is filled, quickly freeze it in liquid N2.
15) Store cells at -80C.

Competency Check:

1) Add 1uL of BBa_J04450 to 300uL of competent cells. Update based on Competent Cell Test Kit provided in your year by iGEM.
2) Incubate on ice for 10 mins.
3) Heat shock for 2 mins at 42C.
4) Spread 30uL of cells on LB+Amp plate .
5) If you can see 100 colonies, then competency is 1x10^9/ug DNA for 300ul E. coli; usually you can get at least 0.1-1x10^8/ug competency.

Flow Cytometry for GFP Expression Measurement

Protocol for Measuring GFP Expression from Flow Cytometry Center at UVA

1) Grow an overnight culture of bacteria of interest. Always grow a negative control culture also that has no GFP and a positive control that has GFP. 2) From overnight culture, inoculate a 1000 uL into 4 mL of LB. 3) At OD~0.4, take first sample. This will be t = 0 mins. Take 350uL from culture and inoculate into 650uL of LB Label tubes properly - VERY IMPORTANT. Place sample in 4C fridge. 4) Take a sample every 60 minutes for the next 6 hours and repeat step 3a & 3b each time.
5) ~60 mins before flow cytometry appointment, take cells out of 4C fridge.
6) Spin down cells for 2 minutes at 8,000 rpm.
7) Resuspend pellet GENTLY in 1 mL of PBS (DO NOT VORTEX).
8) Spin down cells again for 2 minutes at 8,000 rpm.
9) Resuspend pellet gently again in 1 mL of PBS (DO NOT VORTEX).
10) Transfer samples from eppendorf tubes to flow cytometry tubes and transport on ice to the center.
11) Collect flow cytometry measurements with at least 40,000 events for each run.

Transformation

This protocol is optimized for DH5a E. Coli Competent Cells.

Materials

• DH5a competent cells
• Plasmid of interest
• LB agar plates with reperspective antibiotic
• LB media
*Before starting set water bath to 42C

Procedure

1) Thaw competent cells on ice. Transfer 100 uL of competent cells into separate tube and add 1ul of plasmid.
For ligation reactions, add 2-4 uL of plasmid. Pipet up and down to mix thoroughly.
2) Incubate on ice for 15-20 minutes.
3) Heat shock cells in water bath at 42C for 90 seconds.
4) Immediately place cells back on ice for 4 minutes.
5) Add 900ul of LB media to cells and grow them out in the shaker at 37C for 1 hour. For kanamycin and ampicillin resistant plasmids, the cells can be grown out for a shorter time.
6) Spin down cells at 8000 rpm for 2 minutes. Discard 800ul of supernatant.
7) Resuspend the cell pellet in the remaining 200 uL of media left in the tube.
8) Plate 150 uL of cells in one plate and 50 uL of cells on another plate. Sterilize spreader using ethanol and flame. Then let the spreader cool down before using it. Take respective amount of cells and pipette them onto the plate. Use spreader to thoroughly spread the cells around the plate.
9) Incubate at 37C overnight.

Flow Cytometry Experimental Design

Why we chose 6 hour time period with one hour sampling: Data from previous work (Dr. Zargar’s work) showed that 5 hours of observation captured quorum response. We chose to extend the timeframe by an hour to ensure our ability to capture the window of quorum activation and accommodate for slower growth. Samples are taken at 60 minute intervals to assess the effect of population growth on sfGFP expression over time. After consulting Dr. Zargar, we concluded that intervals less than one hour would not show significant change between time points, with one hour better capturing trends.

Why we chose that t = 0 is OD600 0.4: We chose OD600 = 0.4 as our t=0 after consulting Dr. Zargar about the density at which quorum activation begins. At OD600 = 0.4, population density is high enough to reach the quorum activation threshold.

Flow Cytometry

Purpose: To visualize the percent of sfGFP fluorescence in a population and trends in sfGFP expression over time.
Intent: To determine whether our modifications to the lsr operon positively affect population level protein expression over time.
Materials: LB (or similar) nutrient broth, spectrophotometer, PBS buffer
1) Grow an overnight culture of bacteria transformed with a version of the synthetic quorum sensing system. Always grow a negative control culture that has no sfGFP and a positive control that has constitutively promoted sfGFP.
2) From the overnight culture, inoculate 1000 uL into 4 mL of LB.
3) At OD~0.4, take first sample. This will be t = 0 hours. Take 300 uL from culture and inoculate into 700 uL of LB. VERY IMPORTANT- Label tubes properly to avoid mixing time points or test strains with controls. Place sample in 4˚C fridge. Samples are cooled to prevent further growth and protein expression, essentially pausing them at the time point they were taken, but without the possible damage or cell death caused by freezing.
4) Take a sample every 60 minutes for the next 6 hours and repeat step 3a & 3b each time.
5) ~60 mins before flow cytometry appointment, take cells out of 4˚C fridge.
6) Spin down cells for 2 minutes at 8,000 rpm.
7) Resuspend pellet GENTLY in 1 mL of PBS (DO NOT VORTEX).
8) Spin down cells again for 2 minutes at 8,000 rpm.
9) Resuspend pellet gently again in 1 mL of PBS (DO NOT VORTEX).
10) Transfer samples from eppendorf tubes to flow cytometry tubes and transport on ice to the center.
11) Collect flow cytometry measurements with at least 40,000 events for each run.

Flow Cytometry Data Analysis

When analyzing our flow cytometry data, we used a negative control (pSQS alone with no sfGFP containing second plasmid) to set sfGFP gates. Gates are essentially constraints on a graph. Our negative control, pSQS, seemed to autofluorescence a little bit. Figures 1 and 2 show percent sfGFP expression of the negative control cell populations. Ideal the gates would be placed so that Q5 and Q8 (quadrants in the figures) contain all of the natural fluorescence of molecules in sfGFP negative cells so that this data can be used as a blank. In results for our strains with sfGFP containing constructs, any fluorescence that occurs in Q6 and Q7 would be considered sfGFP expression and any fluorescence in Q5 and Q8 would be considered natural autofluorescence that the flow cytometer is picks up even in sfGFP negative cells. We had an unusually high amount of autofluorescence in our negative control. After discussing this is with the experts at the Flow Cytometry Center at the University of Virginia, they suggested that this autofluorescence is likely happening due to debris in the sample or large cell clumps the machine is picking up. They suggested that we use an image cytometer to verify this but due to time constraints, we were not able to complete this in time. Instead, we decided to create liberal gates (Figure 1) and conservative gates (Figure 2). The liberal gates take into account the possible debris in the sample and shift the gate to the left. The conservative gates assume that there is no debris or cell clumping and moves the gates past all the blue dots. We used both gates to analyze our data and compared each to the results we expected based on our model. When using the conservative gates, many of our sfGFP expression levels were below 5%, with some samples showing negative sfGFP expression (below 0%). With this gating, it was difficult to see quorum activation curves. We decided to use liberal gates for our analysis so that trends of quorum activation curves were visible in our data. As a result, our results are more representative of quorum trends with relative sfGFP expression than pure sfGFP expression.

   <img src="" alt="Experiment">
   <figcaption>Figure 1: Liberal Flow Gates </figcaption>

</figure>

   <img src="" alt="Experiment">
   <figcaption>Figure 2: Conservative Flow Gates </figcaption>