Part:BBa_K2243018

Bxb1 attB_322F_Bxb1 attP

To test the influence of Bxb1 attB/P to terminator L3S3P22 (abbreviation: 322) in the forward direction.

Usage

We constructed this part to characterize the recombination efficiency of the recombinase Bxb1 gp35 (BBa_K2243012). It consists of a constitutive promoter (BBa_J23119) flanked by attB and attP sites of recombinase Bxb1 gp35. Different orientation of attB and attP allows the sequence to be flipped, excised, or inserted between recognition sites, which makes it useful for gene editing. Upon recombination, the orientation of the constitutive promoter change. As a result, expression of downstream sequence is shut down, and upstream sequence is transcribed.

Biology

The attP site of Mycobacteriophage Bxb1 is used to integrate phage DNA at the host attB site of Mycobacterium smegmatis, generating the recombinant junctions attL and attR. DNA cleavage and re-ligation occur at the central crossover region at attB and attP, which allows the sequence to be flipped, excised, or inserted between recognition sites. We obtained the terminator, attB and attP sites by oligo synthesis.

Characterization

1. We first characterized the terminator strength using the following formula: Ts=〖[GFP]〗_(random sequence)/〖[GFP]〗_terminator Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 4
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 24
  Illegal BsaI site found at 103
  Illegal BsaI.rc site found at 147