Team:ZJU-China/B Switch

One crucial element we use in GeneSocket is the bistable switch. Bistable switch has been a classical part of genetic modification. First presented in 2000, the original bistable switch was composed of only two repressors and two constitutive promoters, while the promoters and controllable signals could be chosen from any suitable ones^[1]. Using negative feedback mechanism, bistable switches could accomplish bistable gene regulation. Later various derivations of bistable switch emerged and enriched its family. There have been ones increasing sensitivity to switch signal, strengthening the expression of the “ON” side of the switch, or even ones reaching more simplicity.

The bistable switch we chose

Based mainly on the research published in 2012^[2], we adopt bistable switch using serine integrases of bacteriophage origin. Similar bistable switch has also been applied in previous iGEM program (like 2012 KAIST team)The integrase can recognize attB and attP sites on a DNA sequence, invert the sequence between them, and generate two new sites attK and attR. Integrase with corresponding excisionase can recognize the generated sites and do similar operation which can change the sequence to its original state. The required attachment sites for serine integrases are merely around 50bp and the minimal sites for integrase bxb1 and its excisionase gp47 have been defined, which simplifies the system much.

How it works

Left is a brief model of how the adopted bistable switch works. The corresponding bxb1 integrase and gp47 excisionase we use here are Int and Xis. The SET signal, which is the expression of Int solely, can reverse the promoter between attB and attP sites, thus expressing the left hand side of the promoter; the expression of both Int and Xis, can recognize attL and attR, invert the promoter back to its previous orientation and turn on the expression of the right hand side.

Figure.1 Working Mechanism

One problem of the system is that the ratio of Int and Xis could have rather large influences on the flip-flop effectiveness. If the background expression of Xis is relatively high, the SET process may not succeed and if Xis to Int ratio is low, the RESET process may also not accomplish enough, leading to a situation that two different sites exist in one cell. To overcome the problem, we adopt the method presented in the referred paper to add tags to Xis, decreasing its background expression.(see the picture right to get complete design in the page)

Figure.2 Circuit Design

References

[1]Gardner, T. S., Cantor, C. R. & Collins, J. J. Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339-342 (2000).

[2]Bonnet, J., Subsoontorn, P. & Endy, D. Rewritable digital data storage in live cells via engineered control of recombination directionality. Proceedings of the National Academy of Sciences of the United States of America 109, 8884-8889, doi:10.1073/pnas.1202344109 (2012).