Discussion and Future Work

It is obvious that artificial buffer RNA, srlA and ytfJ, works well on comparator, while nanC doesn’t work as expected. It indicates that srlA and ytfJ do have competitive advantages to interact with spot42 against galK::GFP which shows that our design is quite practicable. To our surprising, the areas of complementary pairing sites of srlA and ytfJ predicted by intaRNA range quite narrow which appears to act weakly with spot42. However, the results are just opposite to expectation. It confirms again that prediction of RNAs pairing is not sufficient for artificial RNA designs. Possible reasons may be the facilitation of interactions brought by hfq, the forming of secondary structures which exptremely favourite for sRNA-mRNA interactions.
It also suggests that nanC, without any modifications (like constructing LacZ-fusion), could not act effectively with spot42. Actually it fits quite well with the microarray analysis where nanC gives only 3.5-fold repression rate. It was reported previously that one of nanC-mutant variants when fused with LacZ, displays additional complementary pairing sites with LacZ(predicted by NUPACK)[11], leading to higher repression rate. It is likely that nanC-lacZ fusion brings about some new characters that facilitate the interactions between nanC and spot42. So it shows great potential to further research on reaction mechanisms between nanC-LacZ and spot42 which might throw light on modifying our comparators.

Future Work

Assembly full version ocean-float alarmerAfter fine-tuning of decision-making devices is finished, we will assembly gas vesicle actuator & NP sensor together to bring our whole ideas together. Certainly it will take a long time to arrive to that peak and compatibility is a thorny problem that may obstruct our integration. To solve those problem, we will make every efforts to design feasible schemes according to our model.

Microfluidics-coupled ratio sensor Stochastic noise caused by population heterogeneity is usually the culprit of quantitative designs for bioengineering. To avoid this, single-cell based detections dependent of microfluidics seems a perfect solution for that problem. In addition to this, bio-pixel arrays associated with arsenic-sensitive genetic circuits constructed by Jeff Hasty[12] shed light on constructing biosensors in chips. We have conceived an microarray that can form gradient solutions of both nitrate and phosphate with an orthogonal fashion. In this way, we could detect output signals in several particular intersections that indicate certain ratio of nitrate and phosphate in this area. Provided that we make a standard gradient curve of both P&N concentration, we can measure particular concentration ratio of liquid samples.For example, to measure a certain ratios of samples, we firstly immobilize the cells on the whole areas of microfluidic chip and then locate the certain areas that produce fluorescent peak. After measurement and comparison with the standard curve, The ratio of samples’ concentration can be conversed through specified formula.

Other potential usage Actually our designs of comparator and ratio sensor can be applied to many other fields for biological decision-making processing since they are RNA calculators in essence. With its advantage like low stochastic noise and rapid response, it can be great tools for next-generation synthetic device.

References

1. Masse, E., F.E. Escorcia, and S. Gottesman, Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli. Genes Dev, 2003. 17(19): p. 2374-83.
2. Mutalik, V.K., et al., Rationally designed families of orthogonal RNA regulators of translation. Nat Chem Biol, 2012. 8(5): p. 447-54.
3. G?rke, B. and J. Vogel, Noncoding RNA control of the making and breaking of sugars. Genes & Development, 2008. 22(21): p. 2914-2925.
4. Vogel, J. and B.F. Luisi, Hfq and its constellation of RNA. Nature Reviews Microbiology, 2011. 9(8): p. 578-589.
5. Beisel, C.L. and G. Storz, Base pairing small RNAs and their roles in global regulatory networks. FEMS Microbiol Rev, 2010. 34(5): p. 866-882.
6. Darren, The small RNA, dsrA, is essential for the low temperature expression of rpos during exponential growth in Escherichia coli. The EMBO journal, 1996. 15(15).
7. Man, S., et al., Artificial trans-encoded small non-coding RNAs specifically silence the selected gene expression in bacteria. Nucleic Acids Research, 2011. 39(8): p. e50-e50.
8. Urban, J.H. and J. Vogel, Translational control and target recognition by Escherichia coli small RNAs in vivo. Nucleic Acids Research, 2007. 35(3): p. 1018-1037.
9. Sharma, V., A. Yamamura, and Y. Yokobayashi, Engineering Artificial Small RNAs for Conditional Gene Silencing in Escherichia coli. ACS Synthetic Biology, 2012. 1(1): p. 6-13.
10. Levine, E., et al., Quantitative characteristics of gene regulation by small RNA. PLoS Biology, 2007. 5(9): p. e229.
11. Beisel, C.L. and G. Storz, The base-pairing RNA spot 42 participates in a multioutput feedforward loop to help enact catabolite repression in Escherichia coli. Mol Cell, 2011. 41(3): p. 286-97.
12. Prindle, A., et al., A sensing array of radically coupled genetic 'biopixels'. Nature, 2012. 481(7379): p. 39-44.