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

OVERVIEW

Based on Keasling[1] and Xu’s[2]work, we devoted to exploring of a kind of novel regulation method. Our work was in two steps: exploiting and exploring.
Exploiting new parts and constructed a toolkit for utilization.
Exploring more of this novel regulation method.

EXPLOITATION

We firstly checked several stem-loops which had been experimented before[3] and standardized them to iGEM part registry for further utilization. Then we designed several stem-loops with different folding free energy and tested their regulation effects. Gather those functional stem loops and we got a toolkit.
There are two important components in our testing work, the testing platform——dual-fluorescent reporter system and the designed elements——stem-loops.

We changed different stem-loops in turn into the dual-fluorescent reporter system to test their regulation effects.

Platform——dual-fluorescent reporter system

Figure 1. Dual-fluorescent reporter system

We constructed a dual-fluorescent reporter system as platform to test these parts. (Figure 1)The promoter（BBa_I0500）, RBS with GFP (BBa_I13500)and mCherry(BBa_J06602) are standard parts in the iGEM part registry. The RNase E site between them is from E.coli pap operon, a relative high efficient endonuclease cleavage site validated through experiments.[4] By comparing the expression of the upstream GFP and downstream mCherry, we analyzed the different protection effect of stem-loops.

We measured the quantitative expression on two levels: the transcriptional level and the translational level. On the transcriptional level, we aimed to find if the difference is actually caused by the varied mRNA degradation. Upon the translational level, we explored the possibility and prospect of this post-transcriptional regulation method on practical application.

Elements——Stem-loops

We designed our regulation elements based on 3 parts: loop, stem and a tail ahead with a pair of CG as “sealing nucleotides”, see as figure 2.

Figure 2: Designed stem loop

Loop: Since the loop makes little contribution to the whole free energy, we used the same loop throughout our designs. That is U-U-A-C-A-U-G-A-U-U.

Stem: The stem-loop’s free energy is mainly decided by the pairing stem section. We chose the ratio of "A+U" to "C+G" as 1:1. Because too many C/G may cause the energy too low and hard to adjust, whereas too many A/U are easy to cause terminating effect owing to the structure similarity to rho-independent terminators[5]. Choosing the ratio of 1:1, we can easily adjust the free energy by changing the length of the paring stem. Besides, we try to avoid poly-U structure on the flanking sequence to avoid terminating effect.

"Sealing nucleotides": A pair of CG at the end of a stem-loop can help stabilizing the secondary structure by avoiding flanking extra base paring in different context circuit.

We evaluated the folding free energy of different stem loops with Mfold web server[6], a convenient on line web server for users. But the prediction may be not accurate enough constrained by the current research of the relationship between structure and free energy. With the growing research on structure prediction, this mechanism will get fully developed.

With the dual-fluorescent reporter system and our designed stem-loops, we validated several different stem loops and developed them into a toolkit, convenient for others’ use with result data measured by us.

EXPLORATION

Based on our experimental design and validation, we go on with further exploring. We modelled the degradation of mRNA when with the interference of stem-loops, after being validated with experimental data, we may realize expression prediction based on given free energy. Click here for more details.

Moreover, with our modelling result, we constructed a software to further explore the relationship between the two. We built a stem-loop database with our toolkit and other potential functional stem-loops uploaded by users. We can conversely export free energy even sequence based on the required expression ratio based on our modelling prediction. On the other hand, given our limited experiments and the constrained development of structure prediction, our model and software still needs to be improved. Click here for more details about our software.

Figure 3: Software page

REFERENCE

[1]. Pfleger, B.F., et al., Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat Biotechnol, 2006. 24(8): p. 1027-32.
[2]. Xu, C., et al., Cellulosome stoichiometry in Clostridium cellulolyticum is regulated by selective RNA processing and stabilization. Nat Commun, 2015. 6: p. 6900.
[3]. Smolke, C.D. and J.D. Keasling, Effect of gene location, mRNA secondary structures, and RNase sites on expression of two genes in an engineered operon. Biotechnol Bioeng, 2002. 80(7): p. 762-76.
[4]. McDowall, K. J., Lin-Chao, S., & Cohen, S. N. (1994). A+ U content rather than a particular nucleotide order determines the specificity of RNase E cleavage. Journal of Biological Chemistry, 269(14), 10790-10796.
[5]. Yarnell W S, Roberts J W. Mechanism of intrinsic transcription termination and antitermination[J]. Science, 1999, 284(5414): 611-615.
[6]. Zuker, M., Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research, 2003. 31(13): p. 3406-3415.