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 dimerization binds to pRpa promotor and activate the downstream LasI's translation.
• 4、LasI protein translation.
• 5-1 5-2、LasI protein catalyze the reaction – substrates in E.coli convert into Las molecules.

To demonstrate 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 standard sample of Las molecule (3OC12) from [http://www.adipogen.com/ Adipogen].
• b) LasI product from BBa_K2315033.
• c) Blank control.
• d) LC-MS result of LasI product from BBa_K2315033.

According to figure 1, we can conclude that our LasI coding sequence have the function of catalyzing the reaction from substrates to Las molecule (3OC12).

Converter functional verification

As the LasI coding sequence works, next we test our converter’s function. 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 with adding three concentrations of Rpa molecule (inducer). As the Rpa concentration increases, the pike area of product decreases.

However, the product exactly exists.

• b) LC-MS result of converter product.

In conclusion, converter have the function of convert Rpa molecule into Las molecule.

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.

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 the downstream of GFP expression, then we can measure the fluorescence of GFP which can reflect the strength of inducer. For this measurement, we design the following experiment: we set two samples, the one was added Rpa molecules and converter bacteria as the experimental group (red line in figure 5), the other one only was added Rpa molecules as the control group (blue line in figure 5). Furthermore, we also wanted to know whether the Rpa molecule concentration influence the translation of GFP, we set 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 by Las molecule, however, Rpa can’t induce GFP’s translation, which means that there won’t have crosstalk between Las and Rpa. Finally, a higher concentration of Las molecule can induce higher GFP’s expression. In a word, Las molecule does have the biological function for signaling 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