Part:BBa_K2315046
Contents
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.
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.
- 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 2, 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.
- 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.
- 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.
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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 85
Illegal NheI site found at 108 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 571
Illegal XhoI site found at 206 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 214
Illegal NgoMIV site found at 278
Illegal NgoMIV site found at 563
Illegal NgoMIV site found at 651
Illegal AgeI site found at 1335 - 1000COMPATIBLE WITH RFC[1000]
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