Discussion

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

Fig. 1 This is aTc induction experiment of K737039. Production of spot42 is induced by aTc. The translation of galK::GFP will be inhibited by spot42 in our expectation. And experimental results show that aTc induction works well for inhibition of GFP expression which is represented with decreasing RFU.

Fig. 2 This is experimental control for IPTG induction to verify whether IPTG induction work as expected. Because of the deletion of Plac, IPTG won’t exert effect to spot42 production which means that spot42 will be constitutively expressed at relatively high levels which repress expression of GFP. To compare with Fig. 1, it is obvious that spot42 gives approximately 2-fold repression rate of galK::GFP expression.

Fig. 3 The GFP expression in all genetic circuits without any induction is shown. K737051 is lacI-repressed GFP generator. R0011-E0240 is PlacI GFP generator without tetR repression. K737038 is IPTG-inducible galK::GFP generator without IPTG induction. K737040 has been mentioned in Figure 2.

Fig.4 aTc induction experiment of BBa_K737041 is shown in (A). IPTG induction experiment of BBa_K737040 is shown in (B). Both of them serve as the experimental control for comparators and ratio sensors. Two genetic circuits when separately introduced into cells won’t respond to the inducers for LacI generator assembled with Ptet while tetR generator with Plac. The experimental results meet our expectation and pose no negative effect to GFP expression. Besides, high concentration of aTc induction to K737039 is closed to the result of (A), which indicates the effects of aTc induction and repression of spot42 to GFP expression.

Fig.5 (A) is IPTG induction experiment of K737052 which is aTc-inducible GFP generator. The genetic circuit is involved in the low copy plasmid pSB4A5 . (B) the experimental control. We can clearly to see that aTc induction of spot42 works perfectly well compared to (B).

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.