Part:BBa_K2315033
Contents
Las molecule generator
Group: Shanghaitech iGEM 2017
Introduction
In synthetic biology, quorum sensing system (QS system) has been studied as a way of bacteria communication. A complete system always includes two parts: a generator of AHL molecules and a reporter which can receive molecules and activate the downstream gene expression. In general, the AHL generator always needs to be induced by other molecules (IPTG、aTc……), then after centrifuging the cell culture, the supernatant will contain both inducer and AHL molecules. If these supernatants are added directly into the reporter, the presence of two types of small molecules may cause unpredictable results. Thus, we need a kind of generator that does not require any inducer. According to the results of Interlab Study this year, we have proved that the constitutive promoter BBa_J23100 is strong enough to induce downstream gene expression. So we constructed a new plasmid by using BBa_J23100, BBa_B0034 and LasI coding sequence BBa_K2315100.
As figure 1 shown above, it demonstrates a simple mechanism: the constitutive promotor induces the expression of downstream LasI protein. LasI protein works as an enzyme to convert other substrate molecule to Las molecule (3OC12), which can freely diffuse out of E.coli and can be collected in the supernatant after centrifugation. To characterize this part, we did a series of experiments:
Generator functional verification
Firstly, for detecting the Las molecule accurately, we used HPLC and LC-MS.
- a) The standard sample of Las molecule (3OC12) from [http://www.adipogen.com/ Adipogen].
- b) LasI product.
- c) Blank control.
- d) LC-MS result of LasI product.
According to figure 2, we can conclude that our Las molecule generator has the function to produce the enzyme and catalyze the formation of Las molecule. (3OC12).
Las molecule standard curve
After detection of the generator’s product, we want to know whether the HPLC data is accurate and reliable. Thus we use the Las molecule bought from Adipogen to make standard samples for testing.
- a) HPLC results for 4 different samples with a Las molecule concentration gradient. The relative peak area shows the amount of Las molecule.
- b) Standard curve made with Origin.
- c) LC-MS result of LasI product
According to the standard curve above, the R^2 value is close to 1, which means that the curve fitting is successful. In conclusion, we can analyze the amount of Las molecule by HPLC with a standard curve.
Las molecule attenuation
If Las molecule can be generated, we would like to know: is it robust? What’s its half-life? To verify its stability, we did the following experiment. Firstly we collected the supernatant of Las molecule generator BBa_K2315033, then we set a time-course from 1h to 7h. Finally we tested these samples by HPLC and LC-MS.
- a) With time increasing, the relative pike area decreases slowly. All the values have the same magnitude (10E8), which means that Las molecule is quite stable in cell culture medium.
- b) LC-MS analysis of converter supernatant in 1h.
So Las molecules are robustly generated and do not attenuate in medium.
Reporter's GFP expression under fluorescence microscope
To demonstrate GFP expression of Las molecule reporterBBa_K2315034, we used fluorescence microscope to observe GFP fluorescence. Figure 5 shows two different samples – one was added the supernatant of Las molecule generator and another wasn’t. We can clearly observe the difference of fluorescence – the positive control has more intense GFP signal than the negative control in the dark field. Thus, Las molecule reporter can work well to test Las molecule.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 284
- 1000COMPATIBLE WITH RFC[1000]
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