Difference between revisions of "Part:BBa K1132003"

Latest revision as of 10:12, 14 November 2013

AND gate with recombinases switching gene regulatory sequences and ORF

DO NOT USE this part! The FimE sites did not work well, this design can be use by replace K137008 by K1077001 and K137010 by K1077000.

This AND gate was built with one promoter surrounded by the PhiC31 integrase sites and one coding sequence surrounded by the FimE sites. To transcript a gene, a promoter has to be in front of the coding sequence, we need to have the promoter AND the coding sequence in the same way. It is an AND gate. In the basic state, the promoter and the gene are in the wrong way. The transcription of the output gene will occur in presence of the both recombinases, when the promoter and the gene are in the right way.

The input signals for this gate are the production of either one or both recombinases PhiC31 and FimE. The output can be choosen at will by insering between the recombinases sites of FimE any ORF containing an RBS site. To insert the reading fram, two restriction sites have been placed between the both FimE recombinases sites, BamHI and ClaI. We also designed a test Biobrick of the gate (BBa_K1132034) 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). In the 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_K1132037).

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

The same type of design was used to build a XOR gate (BBa_K1132004).

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.

Sequence and Features