Team:Rice/Experiments

Hexatri

WETLAB EXPERIMENTS

Outline

  1. Determining Chromate Reduction by Reductase: 1,5-Diphenylcarbazide derivatization with potassium chromate assay
  2. Assessing Chromate Transport by Sulfate Transporter System: cysPUWA transformant growth assay with chromate
  3. Assessing Fail-Safe "Kill Switch" Mechanism: IPTG induction of lac-I repressed promoter with fluorescent reporter mCherry

I. DPC Assay

A 1,5-diphenylcarbazide colorimetric 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 amount of Cr(VI) in the solution when interfering substances (such as molybdenum, vanadium, and mercury) are not present.

We used a DPC assay to compare the Cr(V) reduction efficiency of E. coli, E. coli transformed with a constitutively expressed reductase (chrR6 or nemA), and E. coli co-transformed with a sulfate transporter system (cysPUWAsbp) and a constitutively expressed reductase (chrR6 or nemA). Comparing the relative efficiencies will allow us to determine if our reductases reduce Cr(VI) beyond native E. coli levels and if reduction is limited by cell permeability to chromate. View the results of the DPC assays here.

Figure 1: 1,5-diphenylcarbazide forms complex with Cr(VI), producing magenta color, which can be quantified by measuring absorbance at 540 nm.

Protocol

Protocol modified from [2]

  1. 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.
  2. 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)
  3. 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

Figure 2: 1,5-Diphenylcarbazide (DPC) assay with varying concentrations of potassium chromate.

Figure 3: DPC standard curve

II. cysPUWA Growth Assay

To assess the effect of cysPUWAsbp membrane sulfate transport proteins on cell membrane permeability to chromate, we used growth rate measurements to obtain data on the cells’ susceptibility to chromium toxicity. We expected that cells overexpressing the sulfate transport system would exhibit slower growth in the presence of chromium compared to wild-type E. coli MG1655 due to higher amounts of toxic Cr(VI) transported into the cell. To separate growth inhibition triggered by chromium toxicity from that caused by metabolic burden of membrane protein overexpression, we tested the following cell cultures during 12 hours of incubation:

  1. MG1655 transformed with cysPUWA construct in LB media with varying concentrations of potassium chromate (20-160 uM)
  2. MG1655 transformed with cysPUWA construct in LB media with no potassium chromate
  3. Wild-type MG1655 in LB media with varying concentrations of potassium chromate (20-160 uM)
  4. Wild-type MG1655 in LB media with no potassium chromate

See our Results page for the outcomes of the growth assay.

Protocol

  1. Perform a serial dilution to prepare potassium chromate in LB samples
    • Added 20 ul of each solution to 180 ul of liquid culture to generate final concentrations of 0 uM, 5, 10, 20, 40, 80, 100 and 160 uM. Therefore, we prepared chromate samples with concentrations of 0, 250, 500, 1000, 2000, 4000, 5000 and 8000 uM.
  2. Dilute all of the liquid cultures with LB to OD600=0.05
  3. Add 180 uL of each of the cultures to the appropriate well in a 96-well plate according to the layout shown in Figure 4
  4. Add 20uL of the appropriate chromate solution to each of the wells according to the plate layout below
  5. Take OD600 measurements at times: 0, 3, 6, 9 and 12 hours.

III. 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. We chose to use a fluorescent reporter protein instead of the endonuclease BamHI in our initial testing because using the "toxin" would require constantly culturing the cells in chromate in order to keep them alive. Results demonstrate different induction levels at varying IPTG concentrations.

Protocol

  1. 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
  2. Measure the OD of the cultures and dilute with media so that all the cultures are the same OD initially
  3. 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
  4. 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).

  5. Prepare an IPTG standard curve by preparing variably concentrated IPTG samples of just media
  6. 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
  7. 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.

References

[1] Method 7196A: Chromium, Hexavalent (Colorimetric). EPA. Web.

[2] Robins, Katherine J. et al. “Escherichia Coli Nema Is an Efficient Chromate Reductase That Can Be Biologically Immobilized to Provide a Cell Free System for Remediation of Hexavalent Chromium.” PLoS ONE (2013): n. pag. Web.