Difference between revisions of "Part:BBa K1132004:Design"

Revision as of 03:12, 5 October 2013

XOR gate with recombinases switching gene regulatory sequences and ORF

Design Notes

This XOR gate was built with one promoter and one restriction sites BamHI surrounded by the PhiC31 integrase site and the FimE sites. The idea is to insert an inverting gene between the recombinase sites with the help of the restriction site. To transcript the gene, the gene will need to be in the right way, to have been switched one time only by one of the both recombinases. This is a XOR gate.

The input signals for this gate are the production of either one or the both recombinases PhiC31 and FimE. The output can be choosen at will by insering between the recombinases sites any ORF containing an RBS site. We also designed a test Biobrick of the gate (BBa_K1132036) with an inverted RFP protein as output.

This gate can be used in any regulation system, provided that the recombinases are assembled following the promoter of your choice with your specific regulations requirements. For example, if you want to activate the gate in presence of aTc and AHL, you just have to put the recombinase after the promoter activated by LuxR/AHL (BBa_R0065) and the promoter activated by aTc under the repression of TetR (BBa_R0040).

Even a relatively small amount of recombinases can switch the DNA fragments. Therefore, it is really important to control the recombinases expression with a well-locked promoter. You can look at our specially designed regulation sequence (riboregulator) to get as low as possible any undesired expression and production of the recombinases (BBa_K1132005, BBa_K1132006, BBa_K1132007, BBa_K1132008, BBa_K1132042) present design, because of the promoter, the polymerase PolT7 is needed to express the gene. This promoter have been used, in order to have an higher level of expression, it can be assimlar to a amplificator. It is why we design one parts with the gate, the RFP inverted and the polymerase T7 after a promoter (BBa_K1132038).

Resetting the gate to its basal state requires a series of excisases capable of switching back the sequences to their native state.

In the same design, we build a XOR gate (BBa_K1132003).

Furthemore, if the gene is inserted inside the gate in the forward direction, the gate will not be an AND gate anymore, but it will only be activated in the presence of PhiC31 and in the absence of FimE.

Source

later

References

[http://www.ncbi.nlm.nih.gov/pubmed/23539178 Amplifying genetic logic gates. Bonnet et al.]
[http://www.ncbi.nlm.nih.gov/pubmed/23396014 Synthetic circuits integrating logic and memory in living cells. Siuti et al.]