Part:BBa_K1433011

Terminator-RFP-RBS-attB-P-attP-RBS-GFP-Terminator

   This part is a circuit which designed to test function of Bxb1 gp35. Bxb1 gp35 is a serine integrase in Mycobacterium phage Bxb1. This intergrase along can typically catalyzes site-specific recombination between attB and attP, the attachment sites on the phage chromosome and host chromosome. This recombination result in reverse of the sequence between attB and attP, then form new sites attL and attR. 

This part is composed of 7 elements.

   1.Terminator(reverse): BBa_B0015, a strong double terminator.
   2.RFP(reverse): J06504, a monomeric RFP optimized for bacteria.
   3.RBS: BBa_B0034, a strong RBS.
   4.attB and attP sites: Recognition site for Bxb1 gp35, Mycobacterium Phage Bxb1 DNA integrase.
   5.Promoter: BBa_J23110, a middle-ground Bacterial constitutive promoter.
   6.GFP: BBa_E0040, green fluorescent protein derived from jellyfish Aequeora Victoria wild-type GFP.
   7.Terminator: BBa_B0015, a strong double terminator.
   This circuit can not express Bxb1 gp35 nor work along. When this part work with BBa_K1433014 or BBa_K1433015, which can express Bxb1 gp35. Bxb1 serine integrase gp35 will mediate reverse of the promoter between attB and attP sites, then change it to new attL and attR sites.
   There are two reporter gene on this circuit, GFP and RFP. At first, the promoter between attB and attP sites is forward and can promote expression of downstream GFP gene to make bacteria looks green. When co-transform of this part and BBa_K1433014 or BBa_K1433015, gp35 will express to reverse the promoter. The reversed promoter can promote the expression of upstream RFP gene, the color change of bacterial colony or solution can be easily observed.
   Co-transformation of this part and BBa_K1433018 can be used to test the background expression of gp35. BBa_K1433018 contains two BBa_B0015 terminator between two homologous arms to suppress the expression of gp35, The co-transformed bacteria should be pure green. Indeed, there are little bacteria present red or mix of green and red because of background leakage. Lambda red is a Lambda phage derived recombination system, it can recombine dsDNA/ssDNA into different kinds of DNA molecules as long as each side of the donor dsDNA/ssDNA are flanked by 36-50bp homologous arms. Lambda red mediate  homologous recombination can replace two terminators with other sequence or gene to insert exogenous genes and gp35 depression make the color change of green to red. Measuring this leakage of gp35 is significant in recombination experiment cause the real reverse ability of gp35 should get rid of interference of background leakage.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 933
  Illegal NheI site found at 956
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 880
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 900
  Illegal BsaI.rc site found at 983
  Illegal BsaI.rc site found at 1684