Coding

Part:BBa_K2243014

Designed by: Li Yulong   Group: iGEM17_Peking   (2017-10-23)


TP901-1-RDF fusion protein

binding to attL and attR sites to reset the state of DNA


Usage and Biology

excisionases are able to recognize and bind attL and attR sequences. With the help of excisionases, the state transitions become reversible. Recombination Directionality Factors (RDF) are a kind of protein that achieves reverse flipping to recover the sequence flipped by recombinases. By co-expression of the integrase with the corresponding RDF, or expression of the integrase-RDF fusion protein, recombination between the attL and attR sites can be induced. Thus, the flip-flop can restore to the previous state.

Fig 1. Schematic drawing of RDF mechanism.

Experience

Excisionase characterization To make sure that the sequence reversing process is efficient and complete, we use two strategies: one is constructing the integrase and its corresponding RDF into polycistronic structure, and the other one is to build the fusion protein of integrases and RDFs. We have been trying to improve the recombination efficiency by replacing the expression vector with different replication origins or inducible promoters and changing the RBS sequence before the coding sequence.


Fig 2. Two strategies we used. A. The polycistronic structure composed the integrase and its corresponding RDF. B. The fusion protein structure of integrase with its corresponding RDF.

The method is similar to the integrase test method. We constructed the input plasmid to express integrases and RDFs and the output plasmid, on which the promoter J23119 locates in the middle of the attL and attR sequence. The input plasmid expresses GFP before induction of integrase and RDF. But if the inversion can occur after induction, the output plasmid can express RFP. By comparing the changes in fluorescence types and intensities before and after induction, the efficiency of inversion can be estimated.

We have tested the flipping efficiency of our Integrase-RDF fusion proteins with a group pf RBS. Flipping performance varies with different RBS. For TP901-RDF fusion proteins, recombination seems not obvious in some cases (RBS B0032, B0034, B0035, phiC 10000, Bxb 1000, int2 3000), while in others both flipping efficiency and leakage and significant.

Fig 3.pTac TP901-RDF fusion protein flipping efficiency with ten RBS. Proportion of GFP subset indicates flipping efficiency.


A second test was supplemented. Similar results support that our Integrase-RDF fusion proteins are able to flip the sequence between attL and attR site of the reporter, but much improvement are needed to reduce the leakage as well as increase the efficiency.

Fig 4.pTac TP901-RDF fusion protein flipping efficiency with two RBS. Proportion of GFP subset indicates flipping efficiency.

To conclude, we found that the Integrase-RDF fusion proteins are able to flip the sequence between attL and attR site (indicated by observed GFP subset), although tuning work remains to be done to construct a controllable, functional bio-flip-flop device.

Reference

Brondsted,L., Ostergaard,S., Pedersen,M., Hammer,K. and Vogensen,F.K."Analysis of the complete DNA sequence of the temperate bacteriophage TP901-1: evolution, structure, and genome organization of lactococcal bacteriophages".Virology 283 (1), 93-109 (2001).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1531
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 28
    Illegal AgeI site found at 1486
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1725


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