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

OVERVIEW

To explore this novel regulation method, we have several steps to go:
1. Employed preliminary experiment to test if the differential expression is caused by stem loop.
2. Predicted the protection effect using the native stem loops.
3. Tested the difference between the native and the designed stem loops.
4. Validated the primary relationship of free energy and quantitative expression using designed stem loops with gradient free energy.
5. Further we tested our result in the tri-fluorescent reporter system.

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PRELIMINARY EXPERIMENTS

Stem loops and Terminators

To achieve differentiated expression between up and down stream genes within a polycistron, we need to find out the mechanism that causes the difference. Is it caused by stem-loops? How does it realize? Is it caused by protecting the upstream gene，or by decreasing the downstream one just like terminators?

Escherichia coli rho-independent transcription terminators are characterized by an RNA structure with a GC-rich stem-loop followed by a series of uridine residues, which is exactly similar to our designed stem loops. However, it is supposed that our designed stem loops work through interfering in the degrading process by resisting to the exoribonuclease instead of terminating the downstream gene. Thus, we constructed circuits to test if the expression of the downstream gene was reduced owing to the stem loops. We inserted a stem loop between two reporters contrast to the control one without stem loops in the intergenic region. Then we measured the reporting proteins both on the mRNA and the protein level. The result are as follows:

Figure 3: The figure shows the fluorescent of downstream mCherry in the circuit with or without stem-loop, and there’s no significant difference between them(P=0.01). Error bars indicate s.d. of mean of all sequences.

Gene sequence

We aimed to develop this regulation method into a toolkit that can be applied to other polycistrons, not only the dual-fluorescent system. So we had to make sure that the dual-fluorescent reporter system we constructed didn’t influence our result. In other words, it was the stem-loop itself that generate this kind difference. So we swapped the location of GFP and mCherry and constructed the following circuit to test it.

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NATIVE STEM LOOPS

It is reported that there are several native stem loops that may have effects on its flanking genes, either at the 3’ termini or the 5’ termini[1]. Ergo, we use two native stem loops from R. capsulatus and E.coli[2] with different free energy to preliminary verify that stem loops in the intergenic region can regulate the relative expression of two reporter genes within polycistrons.

Figure 7: The protein fluorescence of upstream GFP to downstream mCherry of different circuits, background subtraction has been normalized with control group.Ratio = {[RFPterm/GFPterm]/[(RFPcontrol/GFPcontrol)mean][3]

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NATIVE VS DESIGNED STEM-LOOPS

By using the native stem loop, we have confirmed that in E.coli, the stem loop at the 3’termini can indeed influence the quantitative expression of its upstream gene. Next we aimed to design nonnative stem loops to verify the precise correlation between the △G and the quantitative expression. But only if this mechanism is determined by △G can we design the stem-loops quantitatively. Thus we need to explore whether the protecting efficiency of the stem loops is determined by its Gibbs free energy or by other factors such as certain specific sequence.

Then we designed 3 stem loops that have the same free energy as a native one (△G=-38.7kcal/mol)[4] but with different base sequence and measured their relative expression of the up and down stream genes on protein and mRNA level.

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THE PRECISE CORRELATION

We designed a series of stem loops of gradient free energy to explore the relationship between free energy and quantitative expression. And measured the relative expression of the up and down stream genes on both mRNA and protein level. The result are as follows:

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FURTHER VERIFICATION

After we got the relationship between free energy and quantitative expression, we wanted to test our result in the tri-fluorescent reporter system.and we constructed the tri-fluorescent reporter system as follows:

The result are as follows:

[1] Carrier, T. A., & Keasling, J. D. (1997). Engineering mRNA stability in E. coli by the addition of synthetic hairpins using a 5′ cassette system.Biotechnology and bioengineering, 55(3), 577-580.
[2] Smolke, C. D., & Keasling, J. D. (2002). Effect of gene location, mRNA secondary structures, and RNase sites on expression of two genes in an engineered operon. Biotechnol Bioeng, 80(7), 762-776. doi: 10.1002/bit.10434
[3] Nojima, Takahiko, et al. "Controlling the expression ratio of two proteins by inserting a terminator between the two genes." Nucleic Acids Symposium Series. Vol. 50. No. 1. Oxford University Press, 2006.
[4] Nilsson, P., & Uhtin, B. E. (1991). Differential decay of a polycistronic Escherichia coli transcript is initiated by RNaseE‐dependent endonucleolytic processing. Molecular microbiology, 5(7), 1791-1799.