Part:BBa_K3202035

Xyls-Pc-Pm-sfGFP

This composite part is a TetR-Pc-tet inducer/promoter system connected with Bxb1, cds for one of three recombinases used in our project.

In our study, this composite part is designed to test out which one among the two promoters, pTet and pBAD, is more rigorous in its functions. Also, it is as well aimed to test out the expression of the recombinase in our system. Scientists discovered that phages Bxb1, TP301, and PhiC31 integrases make use of LSTP integrases in mediating phage integration and excision into the bacterial genome between their cognate recognition sites, attB (bacterium) and attP (phage). By pLacIng these sites in the opposite orientation, LSTP integrases cleave, rotate and rejoin the DNA to invert the region between sites.

As shown in figure 1, LSTP integrases catalyze insertion of phage genome (yellow) into the bacterial genome (blue) between attB and attP sites, which form hybrid attL and attR sites. Multicolored arrowheads illustrate the sequence changes that occur during strand exchange, with the core sequence shown in green. In our team, we include the recombinase in order to implement the permanent memory by inverting the orientation of the intervening PR1 according to article Permanent genetic memory with >1-byte capacity. The recombinase is able to flip the DNA in only one direction and thus implement permanent memory.

When it is applied to our circuit, PR1 is inverted in regular working condition where recombinase catalyze the cleavage of each cognate recognition sites attB and attP, inverting the orientation of PR1 and rejoining the circuit at attL and attR respectively. Due to the fact that recombinase can only flip the DNA in one direction, the inverted PR1 which is inhibited in working condition remains inverted even when the recombinase doesn’t express. When the inducer is absent, R1 doesn’t inhibit the expression of the inverted PR1 anymore, the toxin in the second layer can be then expressed and conduct suicide. The inversion is a permanent effect on the bistable system, that is to say even if we stimulate the whole system again by adding inducer, PR1 will not switch its orientation back.

By seeing what color the system displays, we are able to employ this composite part to test the efficiency of the recombinase system.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 1126
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 993
  Illegal NheI site found at 1016
  Illegal SpeI site found at 1126
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 32
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 1126
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 1126
  Illegal NgoMIV site found at 754
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 217
  Illegal SapI.rc site found at 1165