Part:BBa_K1433011

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

This part is a circuit designed to test the function of Bxb1 gp35. Bxb1 gp35 is a serine integrase of Mycobacterium phage Bxb1. This intergrase can exclusively catalyze site-specific recombination between attB and attP, the attachment sites on phage chromosome and host chromosome. This recombination results in the reverse of the sequence between attB and attP and the formation of new sites attL and attR.

Composition:

1. Terminator (reverse): BBa_B0015, a strong double terminator.
2. RFP (reverse): BBa_J06504, a monomeric RFP optimized for bacteria.
3. RBS: BBa_B0034 BBa_B0034, a strong RBS.
4. attB and attP sites: Recognition sites 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 neither express Bxb1 gp35 nor work along. When this part works with BBa_K1433014 or BBa_K1433015, Bxb1 gp35 can be expressed, which will then mediate the reverse of the promoter between attB and attP, and change it to new attL and attR.

INT+SET
There are two reporter genes on the circuit, GFP and RFP. At first, the promoter between attB and attP sites is in forward direction and can promote the expression of downstream GFP gene to make bacteria green. When the co-transform of this part and BBa_K1433014 or BBa_K1433015 occurs, gp35 will express and then reverse the promoter, which will promote the expression of upstream RFP gene. The change of color of bacterial colony or solution can be easily observed.

INT+SOCKET
Co-transformation of this part and BBa_K1433018 can be used to test the background expression of gp35. BBa_K1433018 contains two BBa_B0015 terminators between two homologous arms, which can suppress the expression of gp35. The co-transformed bacteria should be pure green. Indeed, there are a few bacteria presenting red or mix of green and red because of background leakage. Lambda red is a Lambda-phage-derived recombination system, which can recombine dsDNA/ssDNA into different kinds of DNA molecules as long as each side of the donor dsDNA/ssDNA is flanked by 36-50bp homologous arms. Lambda-red-mediated homologous recombination can replace two terminators with exogenous sequences, thus removing gp35 depression and changing the color from green to red. Measuring this leakage of gp35 is important in recombination experiment because the real reverse ability of gp35 should be under no 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