From 2014.igem.org

Latest revision as of 02:57, 18 October 2014

RNAT·RESULT

Choose the best RNAT

In the <a href="https://2014.igem.org/Team:SYSU-China/content.html#Project/Design/RNAT">RNAT·DESIGN</a>, we have introduced that typical types of RNATs coding for heat shock proteins and virulence factors meet the requirements of temperature control, so we decided to use one common type. Fortunately, the 2008 iGEM team, TUDelft, has modified three kinds of natural zipper-like RNATs: Rose element (for short: ROSE), FourU element (for short FourU) and PrfA element (for short PrfA), which have already been made into basic parts of biobricks (<a href="http://parts.igem.org/Part:BBa_K115001">BBa_K115001</a>, <a href="http://parts.igem.org/Part:BBa_K115002">BBa_K115002</a> and <a href="http://parts.igem.org/Part:BBa_K115003">BBa_K115003</a>). We then construct three plasmids containing these three RNATs respectively, between a constitutive promoter (<a href="http://parts.igem.org/Part:BBa_J23119">BBa_J23119</a>) and a reporter protein-mRFP (<a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a>), transformed the three plasmids into top10, to test whether these modified RNATs work well. We used the existing biobrick (<a href="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a>) in which only the FourU element is replaced by RBS compared to our experimental group, serving as the positive control. According to experiment results done by TUDelft, the temperature threshold of the part we choose is 37°C for FourU and PrfA, and 42 °C for Rose. So we set the culture temperature to 28°C, 37°C and 42°C, and if the RNAT work well, we could observe bacteria containing plasmid with FourU and PrfA cultured at 37°C and 42°C and the one with Rose cultured at 42°C changes to red and no change in the bacteria cultured in 28°C, that means these three RNAT change their conformations to the open state so that the ribosome could access the SD sequence.

<img src="" style="width:500px;margin-left:100px"></a> <img src="" style="width:550px;margin-left:75px"></a>

According to the results, only the bacteria containing the plasmid with FourU element cultured at 37°C and 42°C become red, so we choose the best RNAT-FourU element. So the expression of PⅧ could be regulated by FourU element tightly responding to temperature changes.The structure of FourU element is showed in Figure 3.

<img src="" style="width:450px;margin-left:125px"></a>

Further verification the working effect of FourU element as temperature-controlled switch.

For further verifying the working effect of FourU element, we tested the fluorescence intensity of RFP protein to measure the expression level of the reporter protein regulated by FourU element as temperature-controlled switch. So we shake the bacteria containing plasmid with FourU element and the positive and negative control groups in the LB with chloramphenicol and set two culture temperature: 30°C and 37°C. After cultured for 24h, we test the fluorescent of RFP protein at excitation wavelength-584 nm and emission wavelength-607 nm.
<img src="" style="width:700px;margin-left:0px" ></a>

And then, we extend the culture time to 30h and choose 3 time point (6h, 24h, 30h) to test fluorescence intensity of RFP protein expressed by each group, to test the level of leaked expression of RFP protein controlled by FourU element.

<img src="" style="width:600px;margin-left:50px"></a>

Figure 5 shows that, after cultured for 30h, the fluoresce intensity of RFP protein regulated by FourU at 30℃can still maintain a low level compared to the positive control cultured at the same temperature.

According to the results, FourU element works very well and the leak of its control is little. So the expression of PⅧ could be regulated by FourU element tightly when sensing temperature.

Test the response of FourU element to temperature changes from 30°C to 37 °C

After further verifying the working effect of FourU element, we tested the response of FourU element to temperature changes from 30°C to 37 °C, for simulating the 3rd phase of our temperature control process. We cultured both the positive control and experimental bacteria at 30°C for 30h, and change the culture temperature to 37°C, and then test the fluoresce intensity of RFP protein after 1h, 2h and 3h. The result are as follows.

<img src=""style="width:700px;margin-left:0px"></a>

According to the results,after the bacteria shifted from 30°C to 37°C for 3h, the expression of RFP protein raise dramatically, that means FourU element shows a rapid response to temperature changes from 30°C to 37 °C. So under our temperature control, the expression of PⅧ could be rich enough for the generation and release of M13 phage offspring rapidly after the temperature shifted to 37°C.

Construction of inducible mRFP expression with RNAT-fourU element

Considering of the different interaction strength of ligand and receptor in the selection process achieved by B2H system, we construct the plasmid in which mRFP expression regulated by FourU element under an inducible promoter, for using different concentration of inducer to simulate different interaction strength of ligand and receptor. Avoiding destructing the structure of RNAT, we choose the araBAD promoter induced by L-arabinose, whose operon are in the upstream of promoter. In the 2014 distribution of biobricks, we found the parts of the promoter: arabinose C (AraC) with Promoter C (PC) (<a href="http://parts.igem.org/Part:BBa_I13458">BBa_I13458</a>) and PBad (<a href="http://parts.igem.org/Part:BBa_I13453">BBa_I13453</a>) modified by MIT in 2005, and integrated the two parts into araBAD operator (<a href="http://parts.igem.org/Part:BBa_K1333315">BBa_K1333315</a>). And then, we constructed this araBAD operator before FourU element and mRFP. The plasmid is showed in Figure 7. Because we didn't have the plasmid in which RFP protein is regulated by nomarl RBS under PBad, we used the part <a href="http://parts.igem.org/Part:BBa_K577881">BBa_K577881</a> , in which only the FourU element is replaced by RBS, as the positive control.

<img src="" style="width:300px;margin-left:200px"></a>

Exploring the appropriate concentration of L-arabinose inducing the araBAD promoter

We used the positive control BBa_K577881 to explore the appropriate concentration of L-arabinose inducing the araBAD promoter. The results are as follows.

<img src=""style="width:400px;margin-left:125px"></a>

According to the result, we could use the concentration range of L-arabinose from 100μM to 50000μM to simulate the different interaction strength of ligand and receptor.

Testing the plasmid of inducible mRFP expression with RNAT-fourU element

We use 50000μM L-arabinose, the highest concentration, to test inducible mRFP expression with RNAT-fourU element under araBAD promoter. The results are as follows.

<img src="" style="width:700px;margin-left:0px" ></a>

Figure 9 shows that temperature almost has no impact on the expression level of reporter protein regulated by normal RBS, however, to the experimental group regulated by the FourU element, the expression quantity of reporter protein cultured at 30°C induced by 50000μM L-arabinose is dramatically lower than the group cultured at 37°C, and there is no detectable fluorescence intensity of the reporter protein without any L-arabinose. The results show that the araBAD promoter has litte leaked expression and works well with the FourU element.

Then we test the response of the inducible plasmid to the temperature changes from 30°C to 37°C. We cultured both the positive control and experimental bacteria at 30°C overnight and changed them to 37°C, and then test the fluoresce intensity of reporter protein after 1h, 2h and 3h. The result are as follows.

<img src="" style="width:400px;margin-left:150px"></a>

We can see from figure 10 that when the concentration of L-arabinose is 50000μM, the inducible expression of reporter protein regulated by the FourU element cultured is significantly higher than the group cultured at 37°C, and there is almost in difference between the two temperature in the positive control. The inducible inducible mRFP expression with RNAT-fourU element shows a repaid response to temperature changes from 30°C to 37°C. According to the result, we could use this inducible plasmid with different concentration of L-arabinose to simulate different interaction strength of ligand and receptor. And this induction experiment for simulation is undergoing.

Integration with B2H system

At present, the integration of the promoter or2, used in B2H system, and our FourU element have already been integrated, you can see the test experiments and results at the part:<a href="https://2014.igem.org/Team:SYSU-China/content.html#Project/Result/Integration">Integration</a>.