WETLAB
Experiments
IPTG Induction and Fluorescence Measurements
To test the failsafe mechanism of the kill switch, we measured fluorescence produced by mCherry under control of Ptrc-2 LacI-repressible promoter over the course of 14 hours. Results demonstrate different induction levels at varying IPTG concentrations.
Protocol
- Prepare liquid cultures of bacteria transformed with IPTG inducible components
- Example: Ptrc promoter with mcherry fluorescent protein construct is expected to produce more fluorescent protein upon addition of IPTG
- Measure the OD of the cultures and dilute with media so that all the cultures are the same OD initially
- Split cultures into the number of IPTG induction concentrations you’d like to test
- Example: if testing 0mM, 0.01mM, 0.1mM and 1.0mM concentrations of IPTG, split each culture into 4 volumes
- Add appropriate amount of IPTG stock to each sample to generate cultures with varying concentrations
- Example: 4 mL of liquid culture were prepared and subsequently split to evaluate the 4 concentrations of IPTG listed in step 3a to give (4) 1 ml samples for the culture. No IPTG was added to the first sample, 0.1uL of 100mM stock was added to generate 1mL* of 0.01mM culture, 1ul of 100mM stock was added to generate 1ml* of 0.1mM culture, and 10ul of 100mM stock was added to generate 1ml* of 1.0mM culture.
(*Volumes of stock added are small enough to be considered negligible).
- Example: 4 mL of liquid culture were prepared and subsequently split to evaluate the 4 concentrations of IPTG listed in step 3a to give (4) 1 ml samples for the culture. No IPTG was added to the first sample, 0.1uL of 100mM stock was added to generate 1mL* of 0.01mM culture, 1ul of 100mM stock was added to generate 1ml* of 0.1mM culture, and 10ul of 100mM stock was added to generate 1ml* of 1.0mM culture.
- Prepare an IPTG standard curve by preparing variably concentrated IPTG samples of just media
- Place the standard curve and culture samples in a 96 well plate and grow at 37C and shaking in TECAN plate reader for 14 hours. Take fluorescence* and OD measurements over the time course
- If using a fluorescent reporter protein. For example, if using mcherry, take excitation/emission measurements at 587/610nm
- Construct a standard curve to obtain the relationship between chromium concentrations and absorbance values by plotting the absorbance measured before incubation as a function of known Cr(VI) concentrations.
DPC Assay
1,5-diphenylcarbazide assay is a standard EPA method for measuring concentrations of Cr(VI). This method can be used to detect concentrations of hexavalent chromium from 0.5 to 50 mg of Cr(VI) per liter [1]. Color change caused by a formation of Cr(VI) - DPC complex provides an accurate way to measure the amounts of chromium in the solution when no interfering substances (molybdenum, vanadium, and mercury) are present.
We used DPC assay to compare chromium reduction efficiency of chrR6 and nemA reductases and to determine if the rate of chromium reduction increases for E. coli cotransformed with chromium reductase overexpression vector and cysPUWA system. View the results of the DPC assays here .
Protocol
Protocol modified from [2]
- Prepare DPC coloring solution
- Prepare 0.1% DPC stock solution by dissolving 125 mg of DPC in 25 mL of acetone
- Prepare color-developing solution by adding together DPC solution to the concentration of 0.01%, H2SO4 to the concentration of 0.1 N and water to the desired volume.
- Prepare cell cultures with Cr(VI) in 96-well plate
- Measure OD600 of seed cultures
- Dilute cultures to the same initial OD (suggested = 0.05).
- Prepare CrVI stock solution of desired concentration by dissolving solid potassium chromate in water
- Prepare serial dilutions of chromium stock solution in a 96 well plate using multichannel pipette
- Add cultures to the 96 well plate
- Incubate for 12 hours at 37 C in a shaking incubator (800 rpm)
- Measure absorbance of the solutions
*Note: Take absorbance measurements at time 0 (after adding chromate) and time 12 hours.
- Pellet the cells by centrifuging the 96-well plate at maximum speed for 15 minutes.
- Add 50 uL of the supernatant to 950 uL of color-developing solution in a separate 96-well plate. Pipette up and down to mix.
- Transfer 200 uL of solution from each well to a 96-well plate for absorbance readings
- Using a TECAN, perform absorbance measurements at 540 nm