Difference between revisions of "Part:BBa K4656008"
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<p>b.Fluorescence intensity of PpchA-pchA-PLEE1-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate</p> | <p>b.Fluorescence intensity of PpchA-pchA-PLEE1-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate</p> | ||
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We introduced the plasmid pet28a-PpchA-pchA-PLEE1-CI-Plam-EGFP into E. coli to reach a reverse inductive effect of butyrate and performed co-culture experiments with different butyrate concentrations. This time, we found that the higher the butyrate concentration, the lower the fluorescence intensity, in line with our expectations, demonstrating the feasibility of our route (Figure 2). | We introduced the plasmid pet28a-PpchA-pchA-PLEE1-CI-Plam-EGFP into E. coli to reach a reverse inductive effect of butyrate and performed co-culture experiments with different butyrate concentrations. This time, we found that the higher the butyrate concentration, the lower the fluorescence intensity, in line with our expectations, demonstrating the feasibility of our route (Figure 2). | ||
Revision as of 16:58, 16 September 2023
pchA-PpchA-PLEE1-CI
Usage and Biology
We verified the feasibility of the receptor module pathway, that is, whether engineered bacteria can sense the rise in butyrate concentration. Using components BBa_K4656001, BBa_K4656004, and BBa_K4656005, we designed a gene route PpchA-pchA-PLEE1-EGFP, and cloned it into the target vector pET28a, then transformed the engineered bacteria. The route formed by these components can be used to verify the feasibility of the sensing module. In the experiment, sodium butyrate of 0mM, 10mM, and 20mM was added respectively, and the fluorescence intensity (OD480/OD600) was measured at 0, 4, 8, 12, 16, 20, 24, and 28h, respectively. We found that 20mM sodium butyrate had the most obvious promoting effect on fluorescence expression, followed by 10mM sodium butyrate, and the addition of 0mM sodium butyrate had almost no increasing effect on EGFP expression even if the observation time was long. Thus, the feasibility of our receptor module pathway was verified, that is, the increase of butyrate concentration could indeed induce the increase of label protein expression of engineering bacteria, that is, our engineering bacteria could indeed feel the increase of butyrate concentration more sensitively.
Then we added an inhibitory protein CI after the genetic route PpchA-pchA-PLEE1-EGFP to reverse the inductive effect of butyrate. We used the components BBa_K4656001, BBa_K4656004, BBa_K4656005, and BBa_K4656006, and designed a gene route PpchA-pchA-PLEE1-CI-Plam-EGFP. It was cloned into the target vector pET28a(+) and then transformed into the engineered bacteria. Among them, CI protein was used to bind promoter Plam and inhibit downstream EGFP expression, and the amount of CI protein produced was reflected by fluorescence intensity. In the experiment, sodium butyrate of 0mM, 5mM, 10mM, and 20mM was added respectively, and the fluorescence intensity (OD480/OD600) was measured at 0, 4, 8, 12, 16, 20, 24, and 28h, respectively. We found that the fluorescence expression at 0mM sodium butyrate was the largest and that at 20mM sodium butyrate, even if the observation time was long, there was almost no effect of increasing the fluorescence expression, and the higher the concentration of sodium butyrate, the more significantly the fluorescence expression was inhibited (indicating the more CI protein production). This once again verified the feasibility of our receptor module pathway -- the increase in butyrate concentration could indeed induce the increase in the expression of the engineered bacteria label protein CI, that is, it verified that our engineered bacteria could indeed feel the increase in butyrate concentration more sensitively.
Experimental results
We introduced the plasmid pet28a-PpchA-pchA-PLEE1-EGFP into E. coli and performed co-culture experiments with different butyrate concentrations. We found that the higher the butyrate concentration, the stronger the fluorescence intensity, demonstrating the feasibility of our route (Figure 1).
a.Fluorescence observation of PpchA-pchA-PLEE1-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate through fluorescence microscopic
b.Fluorescence intensity of PpchA-pchA-PLEE1-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate
We introduced the plasmid pet28a-PpchA-pchA-PLEE1-CI-Plam-EGFP into E. coli to reach a reverse inductive effect of butyrate and performed co-culture experiments with different butyrate concentrations. This time, we found that the higher the butyrate concentration, the lower the fluorescence intensity, in line with our expectations, demonstrating the feasibility of our route (Figure 2).
a.Fluorescence observation of PpchA-pchA-PLEE1-CI-Plam-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate through fluorescence microscopic
b.Fluorescence intensity of PpchA-pchA-PLEE1-CI-Plam-EGFP engineered bacteria fluid cocultured with different concentrations of butyrate
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 143
Illegal AgeI site found at 609 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 75