Difference between revisions of "Part:BBa K3463019:Experience"

 
 
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how you used this part and how it worked out.
 
how you used this part and how it worked out.
  
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===Applications of BBa_K3463019===
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===Characterizationof BBa_K3463019===
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The purpose of this experiment was to evaluate the correlation between the BHL concentration in the medium and the intensity of the fluorescence emitted.
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To evaluate if our system works, we performed 6 bacterial cultures of transformed <i>E. coli</i> Nissle incubated with increasing amounts of synthetic BHL in the medium from 10nM to 100µM. Once all the media have been set up at T0 with 100µM / 10µM / 1µM / 100nM / 10nM and without BHL, bacteria were inoculated at OD600nm=0.1.Then, fluorescence measurements of eGFP were performed for 15H (hourly in triplicata until 7H) in opaque 96-wells (FluoStars). For each curve in figure 1, the first measurement point (T0) was set at 0 and subtracted to the following ones.
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This experiment was repeated 3 times.
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In addition, a culture of wild type <i>E. coli</i> Nissle (not transformed) was used  as a negative control.
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[[Image:T--Grenoble Alpes--Res Survival BHL fluo.png|600px|thumb|center| '''Figure 1''' eGFP expression according to the BHL concentration and over time. Different concentrations of BHL were performed to evaluate the effect of BHL in <i>E. coli</i> Nissle. Thanks to the BHL, the engineered <i>E. coli</i> should be able to express eGFP. Without BHL, no eGFP expression should be detected.]]
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Averages : 100µM/10µM/1µM > others
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The Figure 1 shows the fluorescence intensity of each bacterial culture according to the BHL concentration and over time. While the wild type <i>E. coli</i> Nissle auto-fluorescence is used as blank, all the other transformed cultures show significant eGFP expression.
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First, we can see that transformed <i>E. coli</i> Nissle produce low quantities of eGFP even without BHL. However, we can also observe that the more BHL, the more eGFP expression (p-value<2.10-16). Indeed, in the culture containing 10µM or 100µM of BHL, the fluorescent intensity starts to increase sharply after 3 hours of culture and reaches 600 or more. Nevertheless, even if  curves reach 400 and 100 with 1µM and 100nM respectively, weaker concentrations of BHL (10nM) don't allow transformed <i>E. coli</i> Nissle to significantly express eGFP.
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Through an ANOVA test, we can conclude that, thanks to the new part BBa_K3463019, our engineered <i>E. coli</i> Nissle is able to sense low concentration of BHL (1µM) in its environment and express a specific gene under the control of PrhlR.
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===User Reviews===
 
===User Reviews===

Latest revision as of 08:41, 27 October 2020


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Characterizationof BBa_K3463019

The purpose of this experiment was to evaluate the correlation between the BHL concentration in the medium and the intensity of the fluorescence emitted.

To evaluate if our system works, we performed 6 bacterial cultures of transformed E. coli Nissle incubated with increasing amounts of synthetic BHL in the medium from 10nM to 100µM. Once all the media have been set up at T0 with 100µM / 10µM / 1µM / 100nM / 10nM and without BHL, bacteria were inoculated at OD600nm=0.1.Then, fluorescence measurements of eGFP were performed for 15H (hourly in triplicata until 7H) in opaque 96-wells (FluoStars). For each curve in figure 1, the first measurement point (T0) was set at 0 and subtracted to the following ones. This experiment was repeated 3 times.

In addition, a culture of wild type E. coli Nissle (not transformed) was used as a negative control.

Figure 1 eGFP expression according to the BHL concentration and over time. Different concentrations of BHL were performed to evaluate the effect of BHL in E. coli Nissle. Thanks to the BHL, the engineered E. coli should be able to express eGFP. Without BHL, no eGFP expression should be detected.

Averages : 100µM/10µM/1µM > others

The Figure 1 shows the fluorescence intensity of each bacterial culture according to the BHL concentration and over time. While the wild type E. coli Nissle auto-fluorescence is used as blank, all the other transformed cultures show significant eGFP expression.

First, we can see that transformed E. coli Nissle produce low quantities of eGFP even without BHL. However, we can also observe that the more BHL, the more eGFP expression (p-value<2.10-16). Indeed, in the culture containing 10µM or 100µM of BHL, the fluorescent intensity starts to increase sharply after 3 hours of culture and reaches 600 or more. Nevertheless, even if curves reach 400 and 100 with 1µM and 100nM respectively, weaker concentrations of BHL (10nM) don't allow transformed E. coli Nissle to significantly express eGFP.

Through an ANOVA test, we can conclude that, thanks to the new part BBa_K3463019, our engineered E. coli Nissle is able to sense low concentration of BHL (1µM) in its environment and express a specific gene under the control of PrhlR.


User Reviews

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