Part:BBa_K4307004

AttP & AttB

AttP and attB are two attachment sites of Bxb1 integrase, each of which is about 50 bp in length. Bxb1 integrase is capable of catalyzing site-specific recombination between two attachment sites (attP and attB) and inverting the DNA sequence flanked by these two opposing sites.

This time, we express Bxb1 integrase in bacteria and check its capability of induce inversion.

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 55
  Illegal BsaI.rc site found at 150
  

Characterization

The following figure demonstrates our successful construction.

Figure 1: The construction results of AttP and AttB determined by sequencing.

Fluorescence assay was done to characterize the biobrick.

In order to test the compatibility of the sequence of two attachment sites we picked with Bxb1 integrase, we placed mCherry and EGFP upstream and downstream of OR1-OR2 promoter respectively.

We incorporated the gene of Bxb1 integrase with pBAD promoter. The fluorescence intensity of mCherry was recorded 16 hours after the E.coli strain was induced with 1% (w/v) arabinose in this trial. The OD600 was also measured in order to standardize the fluorescence signal per cell. Different experiments were induced in one 96 well plate and all samples were separated to two wells to get an average result. The OD600 and fluorescence signal was recorded in a plate reader after 16 hours induction at 37°C or 16°C.

Figure 2: Characterization of serine integrase function by fluorescence spectrophotometry.

Conclusion

We demonstrated that the attP and attB we selected could be recognized by Integrase and trigger inversion, which laid the foundation for the design of logic gating.