Difference between revisions of "Team:Rice/Results"

 
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<h1>Results</h1>
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<p>Here you can describe the results of your project and your future plans. </p>
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<h5>What should this page contain?</h5>
 
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<li> Clearly and objectively describe the results of your work.</li>
 
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<li> Future plans for the project. </li>
 
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<li> Considerations for replicating the experiments. </li>
 
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<h5>You should also describe what your results mean: </h5>
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<h1>RESULTS</h1>
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<h2>Overview</h2>
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<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
 
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<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
 
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<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
 
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<h2>chrR6 Chromium Reduction Activity</h2>
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<p> As detailed on our "Experiments" page, we measured chrR6 chromium reduction activity using DPC assay. The strong r<sup>2</sup> 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.
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<p class = "caption"><b>Figure 1:</b> DPC standard curve <p/>
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<p>Figure 2.A compares the Cr(VI) reduction rate standardized for OD<sub>600</sub> of <i>E. coli</i> MG1655 versus <i>E. coli</i> MG1655 cotransformed with the sulfate transporter system (BBa_K2194004) and constitutively expressed chrR6 reductase enzyme (BBa_K2194000).</p>
  
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<img src="https://static.igem.org/mediawiki/parts/2/2a/Cr_VI_cumulative_reduction1_rice.png" " style="width:50%">
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<p class = "caption"><b>Figure 2A:</b> DPC Assay Reveals that MG1655 <i>E. coli</i> more efficient Cr(VI) reducers above threshold initial  [Cr(VI)] </p>
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<h5> Project Achievements </h5>
 
  
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
 
  
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<li>A list of linked bullet points of the successful results during your project</li>
 
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
 
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<p> Figure 2.B presents the same data as Figure 2.A but as an amount of total Cr(VI) reduced rather than the OD<sub>600</sub> 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.
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</p>
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<div class = "center caption">
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<img src="https://static.igem.org/mediawiki/parts/1/1d/Cr_VI_percent_reduction1_rice.png
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" " style="width:50%">
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<div class = "center">
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<p class = "caption"><b>Figure 2B:</b> DPC Assay Reveals that MG1655 <i>E. coli</i> more efficient Cr(VI) reducers above threshold initial  [Cr(VI)]
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</div>
 
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<p> 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 <i>E. coli</i> 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. </p>
  
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<h5>Inspiration</h5>
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<div class = "center caption">
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<p>See how other teams presented their results.</p>
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<img src="https://static.igem.org/mediawiki/parts/8/84/NemA-chrR6_rice.png
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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<div class = "center">
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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<p class = "caption"><b>Figure 3:</b> chrR6 reductase enzyme is more efficient than nemA reductase in reducing Cr(VI)
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<h2>cysPUWA Effect on Cell Membrane Permeability to Cr(VI)</h2>
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<p> In this experiment, we added varying concentrations of Cr(VI) to 0.05 OD<sub>600</sub> cultures of: <i>E. coli</i> MG1655, <i>E. coli</i> MG1655 transformed with a plasmid constitutively expressing reductase chrR6, and <i>E. coli</i> 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 OD<sub>600</sub> measurements (presented here). The data indicates that the sulfate transporter system introduces toxicity to the cell (because it has the lowest OD<sub>600</sub> values for every initial concentration of Cr(VI) except [Cr(VI)]<sub>i</sub>=0). Because the OD<sub>600</sub> 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. </p>
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<div class = "center caption">
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<img src="https://static.igem.org/mediawiki/parts/1/18/Cys_growth.png" " style="width:60%">
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<p class = "caption"><b> Figure 4: </b> Effect of Chromate on <i>E. coli</i> Growth  </p>
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<h2>PgntK Promoter Activity</h2>
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<p> In this experiment, we compared the reporter protein expression of <i>E. coli</i> MG1655 transformed with a <i>PgntK</i> promoter+mCherry construct versus a <i>Pcon</i> promoter+mCherry construct. The data indicates that <i>PgntK</i> promoter has a comparable transcription initiation rate to <i>Pcon</i> promoter when no membrane stress is present. </p>
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<img src="https://static.igem.org/mediawiki/parts/4/45/Pgntk.png" " style="width:60%">
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<p class = "caption"><b> Figure 5: </b> <i>PgntK</i> (BBa_K2194002) Promoter similar strength to <i>Pcon</i> (BBa_J23108)  </p>
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<h2>Kill Switch Failsafe Mechanism</h2>
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<img src="https://static.igem.org/mediawiki/parts/b/bd/Iptg_induction.png" style="width:50%">
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<p class = "caption"><b>Figure 6:</b> IPTG induces toxin expression leading to cell death <p/>
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<p>
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In this experiment, we transformed <i>E. coli</i> MG1655 with a plasmid containing promoter <i>Ptrc-2</i> and a fluorescent reporter protein mCherry and added varying amounts of IPTG to culture with the same initial OD<sub>600</sub>. 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 <i>Ptrc-2</i> promoter is lacI-repressed as predicted. </p>
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 +
<p>
 +
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 <i>mCherry</i> in our construct for <i>BamHI</i> and measure OD<sub>600</sub> over time in cultures with chromate and varying concentrations of IPTG.
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<img src="https://static.igem.org/mediawiki/parts/1/1d/Rice_iptg_plot2.png" style="width:60%">
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<p class = "caption"><b>Figure 7:</b> IPTG-induced mCherry fluorescence/OD vs. time <p/>
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Latest revision as of 15:31, 16 December 2017

Hexatri

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.

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).

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.

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.

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.

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.

Figure 5: PgntK (BBa_K2194002) Promoter similar strength to Pcon (BBa_J23108)

Kill Switch Failsafe Mechanism

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.

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