Template:Virginia/Experiments

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.

Chemical 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

Flow Cytometry

Purpose: Visualize percent of sfGFP fluorescence in a population.
Intent: To determine whether our modifications to the lsr operon affect population level protein expression.
Materials: LB (or similar) nutrient broth, spectrophotometer, PBS buffer.

Procedure

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 300uL from culture and inoculate into 700uL of LB. Label tubes properly - VERY IMPORTANT. Then 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.