Difference between revisions of "Part:BBa K2315046"

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Rpa-Las signal converter

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. However, there is rarely molecule converter, which can receive a kind of AHL molecule and convert it into another AHL molecule. In our project this year, we pay attention to two kinds of QS systems: Las and Rpa. We successfully construct a ‘converter’– it can receive Rpa molecules and then generate Las molecules. This device achieves a simple logic circuit, moreover, It may lay the foundation for constructing a much more complex logic circuits.

Figure 1. The mechanism of converter

As shown in figure 1, the conversion process includes 5 steps:

• 1、Rpa molecules enter E.coli.
• 2、RpaR protein translation after the induction of constitutive promotor and Rpa promotor (can be induced by Rpa molecules). Then Rpa molecules make

RpaR proteins dimerization.

• 3、The dimerized RpaR binds to pRpa promotor and activate the downstream LasI transcription.
• 4、LasI protein translation.
• 5-1 5-2、LasI protein catalyzes the reaction that converts E.coli substrates into Las molecules.

To demonstrate the function of this part, we did a series of experiments:

LasI (coding sequence) functional verification

Firstly, we test the function of LasI coding sequence. We construct a plasmid BBa_K2315033 which contains a constitutive promotor BBa_J23100, a strong RBS BBa_B0034 and LasI coding sequence BBa_C0178. 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 chromatography of standard sample of Las molecule (3OC12) from [http://www.adipogen.com/ Adipogen].
• b) The chromatography of LasI product generated from BBa_K2315033.
• c) Blank control.
• d) LC-MS analysis of LasI product from BBa_K2315033.

According to figure 2, we can conclude that the LasI coding sequence we use can generate the enzyme to convert substrates to Las molecules (3OC12).

Converter functional verification

As the LasI coding sequence works, next we test the function of this part as a converter. Also by using HPLC and LC-MS, we successfully detected the product from converter.

Fig. 3 HPLC and LC-MS detection of Las molecule (3OC12) from converter BBa_K2315046

• a) Converter product Las molecules are detected with HPLC at three different concentrations of Rpa molecule (inducer). As the Rpa concentration increases, the pike area of product decreases. However, the generation of Las molecules do exist.
• b) LC-MS analysis of converter product.

In conclusion, the converter has the function to convert Rpa molecules into Las molecules.

Las molecule attenuation

If Las molecule can be generated, we should ask a question: 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 to check Las molecule stability. Finally we tested these samples by HPLC and LC-MS.

Fig. 4 Las molecule attenuation

• a) With time increasing, the relative pike area decreases slowly. All the values have the same magnitude (10E8), which means Las molecule is quite stable in cell culture medium.
• b) LC-MS analysis of converter product after 1 hour induction.

In summary, Las molecule can be robustly generated and there is no obvious attenuation in cell culture supernatant.

Converter gradient induction

Now we have done a lot for characterization of Las molecule, how about its biological function? Does it work as an inducer or a signal molecule? Thus we constructed another plasmid BBa_K2315034. This part works as a molecule receiver and reporter – It can be induced by Las molecule and activate downstream GFP expression. We can measure the fluorescence of GFP that reflects the strength of inducer. For this measurement, we did the following experiment: two samples were prepared, one with Rpa molecules and the converter bacteria added as the experimental group (red line in figure 5), the other one with only Rpa molecules added as the control group (blue line in figure 5). Furthermore, we also wanted to know whether the concentrations of Rpa molecule influence the translation of GFP, thus we tested a series of concentrations of Rpa molecule from 10E-9 to 10E-5. From figure 5 we notice that the Las molecule reporter BBa_K2315034 has an obvious response to Las molecule. However, Rpa cannot induce GFP expression, demonstrating no crosstalk between Las and Rpa. Notably, the expression level of GFP increases with increased Las molecule concentration. In a word, Las molecule generated by the converter does have the biological function for signalling and inducing.

Fig. 5 Converter gradient induction with Rpa molecule

Converter's time response

The GFP’s expression must be different among different induction time. We also did an experiment. After a defferent induction time of 3h, 6h and 9h, We added converter’s supernatant to the reporter with a gradient of Rpa molecule concentration for converter’s induction. the curve shows that >6h, the GFP’s expression don’t have apparent difference, however, when time = 3h, the expression is less than the other two times. Not surprisingly, concentration gradient of Rpa molecule and incubation time of Las reporter, these two factors won’t influence each other. To conclude, converter have a time response for Rpa molecule induction.

Fig. 6 Converter 's time response

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 7 shows two different samples – one was added Las molecule generator (BBa_K2315033)’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. 7 Reporter's GFP expression

Sequence and Features