Difference between revisions of "Part:BBa K2315033"

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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.

Figure 1. The mechanism of Las molecule generator

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 use HPLC and LC-MS.

Fig. 2 HPLC and LC-MS detection of Las molecule (3OC12) from generator BBa_K2315033

• 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.

Fig. 3 HPLC and LC-MS detection of Las molecule for standard curve

• 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.

Fig. 4 Las molecule attenuation

• 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.

Fig. 5 Reporter's GFP expression

Sequence and Features