iGEM 2017 — Team Rice

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# Team:Rice/Notebook

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# LAB NOTEBOOK

[May](https://static.igem.org/mediawiki/parts/2/26/Rice_igem_May.pdf)

[June](https://static.igem.org/mediawiki/parts/5/51/Rice_igem_June.pdf)

[July](https://static.igem.org/mediawiki/parts/9/96/Rice_igem_July.pdf)

[August](https://static.igem.org/mediawiki/parts/2/26/Rice_igem_august.pdf)

[September](https://static.igem.org/mediawiki/parts/a/ad/Rice_igem_september.pdf)

[October](https://static.igem.org/mediawiki/2017/9/9f/October.pdf)

[Protocols](https://static.igem.org/mediawiki/2017/9/9a/Protocols_final.pdf)

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# Team:Rice/Experiments

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# 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.](https://2017.igem.org/Team%3ARice/Results)

![](https://static.igem.org/mediawiki/parts/d/d5/Dpc_complex.png)

**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]](http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059200)

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

![](https://static.igem.org/mediawiki/parts/6/6f/DPC_chromate_assay.jpeg)

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

![](https://static.igem.org/mediawiki/parts/d/d0/Dpc_standard_curve.png)

**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)- MG1655 transformed with *cysPUWA* construct in LB media with no potassium chromate
   - Wild-type MG1655 in LB media with varying concentrations of potassium chromate (20-160 uM)
   - Wild-type MG1655 in LB media with no potassium chromate

See our  [Results](https://2017.igem.org/Team%3ARice/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](https://2017.igem.org/Team%3ARice/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.

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# Team:Rice/Results

* [Home](https://2017.igem.org/Team%3ARice)
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* Team
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[Hexatri](#!)

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# RESULTS

## Overview

## chrR6 Chromium Reduction Activity

As detailed on our "Experiments" page, we measured chrR6 chromium reduction activity using DPC assay. The strong r2 value of 0.9944 for the linear trend line of our standard DPC curve indicates that this is an accurate Cr(VI) detection method within this range of concentrations. Therefore, when we investigate the reduction efficiencies of our reductases, we will use initial Cr(VI) concentrations within this range.

![](https://static.igem.org/mediawiki/parts/9/9c/Standard_curve.png
)

**Figure 1:** DPC standard curve

Figure 2.A compares the Cr(VI) reduction rate standardized for OD600 of *E. coli* MG1655 versus *E. coli* MG1655 cotransformed with the sulfate transporter system (BBa\_K2194004) and constitutively expressed chrR6 reductase enzyme (BBa\_K2194000).

![](https://static.igem.org/mediawiki/parts/2/2a/Cr_VI_cumulative_reduction1_rice.png)

**Figure 2A:** DPC Assay Reveals that MG1655 *E. coli* more efficient Cr(VI) reducers above threshold initial [Cr(VI)]

Figure 2.B presents the same data as Figure 2.A but as an amount of total Cr(VI) reduced rather than the OD600 standardized rate. (The x-axis and the light blue bars display the initial concentration of Cr(VI) and the y-axis displays the final concentration of Cr(VI). Comparison of the dark blue bars to the light blue bar at each different initial concentration of Cr(VI) reveals the total change in [Cr(VI)].The data indicates that below an initial [Cr(VI)] of 200 uM the cotransformed bacteria are less efficient at reducing Cr(VI) than wild type bacteria. Above 200 uM initial [Cr(VI)], the trend reverses and wild type bacteria are less efficient.

![](https://static.igem.org/mediawiki/parts/1/1d/Cr_VI_percent_reduction1_rice.png
)

**Figure 2B:** DPC Assay Reveals that MG1655 *E. coli* more efficient Cr(VI) reducers above threshold initial [Cr(VI)]

In the experiment which results are shown below in Figure 3, we compared the Cr(VI) reduction efficiency of chrR6 versus nemA at different initial concentration of Cr(VI). The two reductases were expressed under constitutive promoters in *E. coli* MG1655. Final concentrations of Cr(VI) for each sample were measured at 12 hours after adding chromate. This data shows that the two enzymes have similar reduction efficiencies.

![](https://static.igem.org/mediawiki/parts/8/84/NemA-chrR6_rice.png
)

**Figure 3:** chrR6 reductase enzyme is more efficient than nemA reductase in reducing Cr(VI)

## cysPUWA Effect on Cell Membrane Permeability to Cr(VI)

In this experiment, we added varying concentrations of Cr(VI) to 0.05 OD600 cultures of: *E. coli* MG1655, *E. coli* MG1655 transformed with a plasmid constitutively expressing reductase chrR6, and *E. coli* MG1655 cotransformed with a plasmid constitutively expressing reductase chrR6 and the sulfate transporter system cysPUWA-sbp. We grew the cultures for 12 hours shaking at 37C and took their final OD600 measurements (presented here). The data indicates that the sulfate transporter system introduces toxicity to the cell (because it has the lowest OD600 values for every initial concentration of Cr(VI) except [Cr(VI)]i=0). Because the OD600 values of the reductase and co-transformed reductase/transporter cultures are comparable at time 12 hours for no chromate, this indicates that the additional toxicity from the sulfate transporters is due to increase permeability of the cell to Cr(VI) rather than increased metabolic load of expressing a sythetically engineered plasmid.

![](https://static.igem.org/mediawiki/parts/1/18/Cys_growth.png)

**Figure 4:**  Effect of Chromate on *E. coli* Growth

## PgntK Promoter Activity

In this experiment, we compared the reporter protein expression of *E. coli* MG1655 transformed with a *PgntK* promoter+mCherry construct versus a *Pcon* promoter+mCherry construct. The data indicates that *PgntK* promoter has a comparable transcription initiation rate to *Pcon* promoter when no membrane stress is present.

![](https://static.igem.org/mediawiki/parts/4/45/Pgntk.png)

**Figure 5:**  *PgntK* (BBa\_K2194002) Promoter similar strength to *Pcon* (BBa\_J23108)

## Kill Switch Failsafe Mechanism

![](https://static.igem.org/mediawiki/parts/b/bd/Iptg_induction.png)

**Figure 6:** IPTG induces toxin expression leading to cell death

In this experiment, we transformed *E. coli* MG1655 with a plasmid containing promoter *Ptrc-2* and a fluorescent reporter protein mCherry and added varying amounts of IPTG to culture with the same initial OD600. Time course measurements of mCherry fluorescence (excitation/emission=587nm/610nm) over 14 hours are shown in Figure 7. This graph shows a standard dose-responsive curve, indicating that the *Ptrc-2* promoter is lacI-repressed as predicted.

In this initial test of the Kill Switch we used mCherry instead of the “toxin” BamHI because including the toxin would require culturing our bacteria in chromate to keep them alive. Instead, we measure mCherry fluorescence as an approximate and proportional cell death response to IPTG induction. In a future test, we would substitute *mCherry* in our construct for *BamHI* and measure OD600 over time in cultures with chromate and varying concentrations of IPTG.

![](https://static.igem.org/mediawiki/parts/1/1d/Rice_iptg_plot2.png)

**Figure 7:** IPTG-induced mCherry fluorescence/OD vs. time

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