Difference between revisions of "Team:OUC-China/Proof"

This year, we devoted to the exploitation of new regulatory parts and the exploration of a kind of novel regulatory method.

TEST PREVIOUS STEM-LOOPS

To collect parts and modify as much new parts as possible for broad use, we began with standardization of the existing elements. Several native stem-loops had been discovered to have influence on flank genes expression and had been tested experimentally. However, they were only tested roughly, without quantitatively characterization of stem-loops. We further measured the stem-loops with our dual-fluorescent reporter system (Figure 1), and standardized them submitting to iGEM part registry with detailed description. Click here to see our registried parts.

Figure 1 Dual-fluorescent reporter system

We constructed the dual-fluorescent reporter system to test the regulatory effect of various stem-loops. After inserting stem-loops into the dual-fluorescent reporter system, we measured the relative expression of the upstream gfp and downstream mCherry to test regulatory effect on both transcriptional and translational level. The result we measured the stem-loop of -25.6kcal/mol is as follows:

Figure 2 Relative expression on RNA and protein level with stem-loop of -25.6 kcal/mol (measured by Mfold) contrast to the control group with no stem-loop. The result is the ratio of upstream gfp to downstream mCherry. Error bars indicate s.d. of mean of experiments in triplicate.

Our result shows that the insert of stem-loop may have regulatory effects. Then we did more to explore. We tested another two stem-loops that have been used before and analyzed the results. The tested stem-loops and results are as follows.

Figure 3 Structures of three discovered stem-loops

Figure 4 Relative expression of 3 stem-loops with different folding free energy. Error bars indicate s.d. of mean of experiments in triplicate.

After made sure that the stem loops did have effects on regualtion, Moreover, we standardized the 3 elements and submit them to iGEM parts registry for others’ use.

---

DESIGN OUR STEM-LOOPS

Besides the confirmation of previous discovered stem-loops, we also devoted to exploring new stem-loops designed by ourselves. The relationship between structure and nergy is a tricky problem and we weighed several software and finally we chose Mfold. Below are the result of testing of new designed stem-loops. The new designed stem-loops are as follows:

Figure 5 Structure of three stem-loops designed by ourselves

Figure 6 Relative expression on RNA and protein level with stem-loop of -14.9 kcal/mol (measured by Mfold) contrast to the control group with no stem-loop. The result is the ratio of upstream gfp to downstream mCherry. Error bars indicate s.d. of mean of experiments in triplicate.

Figure 6 Relative expression on RNA and protein level with stem-loop of -30.1 kcal/mol (measured by Mfold) contrast to the control group with no stem-loop. The result is the ratio of upstream gfp to downstream mCherry. Error bars indicate s.d. of mean of experiments in triplicate.

Figure 6 Relative expression on RNA and protein level with stem-loop of -34.4 kcal/mol (measured by Mfold) contrast to the control group with no stem-loop. The result is the ratio of upstream gfp to downstream mCherry. Error bars indicate s.d. of mean of experiments in triplicate.

These parts are also have fine regulatory effects Combine those validated previous stem-loops and our designed ones, we got a set of stem-loops.