Part:BBa_K2315033
Contents
Las molecule generator
Group: Shanghaitech iGEM 2017
Introduction
In synthetic biology, quorum sensing system (QS system) has been researched as a way of bacteria communication. A whole system always concludes two parts: a generator of AHL molecules and a reporter which can receive molecules and activate the downstream genes’ expression. Generally speaking, the AHL generator always need to be induced by other molecules (IPTG、atc……), then after centrifuge the mixture, the supernatant will conclude both inducer and AHL molecules. If we need add it into the mixture of reporter, the inducer may influence the following experiment, moreover, it will occur unknown impacts. Thus, we need a kind of generator which does not need any inducer. According to the results of Interlab Study this year, we have proved that the constitutive prototor BBa_J23100 is strong. So we construct a plasmid by using BBa_J23100, BBa_B0034 and LasI coding sequence BBa_K2315100.
As figure 1 shown above, it has a simple mechanism: the constitutive promotor induce the downstream of LasI protein. LasI protein works as an enzyme to catalyze the reaction from substrates to Las molecule (3OC12), the molecule can freely diffuse out of E.coli and can be collected in the supernatant after centrifuge. To characterize this part, we did a series of experiments:
Generator functional verification
Firstly, for detecting the Las molecule accurately, we use 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 have the function of catalyzing the reaction from substrates to Las molecule (3OC12).
Las molecule standard curve
After detection of 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 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 by 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 Las molecule amount by HPLC with standard curve.
Las molecule attenuation
If Las molecule can be generated, we would ask a question: is it robust? What’s its half-life period? 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 gradient from 1h to 7h. Finally we test these samples by HPLC and LC-MS.
- a) With time increasing, the relative pike area decreases slowly. The all values have the same magnitude (10E8), which means that Las molecule have a
high stability.
- b) LC-MS result of converter in 1h.
So Las molecule has a high stability and very robust.
Reporter's GFP expression under fluorescence microscope
For demonstrating GFP expression of Las molecule reporterBBa_K2315034, we used fluorescence microscope to observe the GFP’s green fluorescence. Figure 5 shows two different samples – one was added the Las molecule generator's supernatant and another wasn’t. Between Negative control and Positive control under dark field, we can clearly distinguish the fluorescence’s difference – Positive control is much brighter than Negative control. Thus, Las molecule reporter can work well for testing 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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