Part:BBa_K1742006

BxBI Reporter (attB:T35S:attP:OmegaUTR)

Bxb1 is a protein from Mycobateriophage Bxb1’s gp35 gen. This integrase is able to recognize two different sites, one in phage’s genome (attP, “Phage attachment site”) and another in bacterial chromosome (attB, “Bacterial attachment site”)[1]. Depending on the position and sense of these sites, bxb1 is able of excising or inverting.

Biology and Usage

In order to see the activity of the integrase, Valencia_UPV 2015 designed the present reporter element (BBa_K1742006) that consists of a Cauliflower Mosaic Virus terminator (T35S) flanked with the recognition sites (attB:T35S:attP:omegaUTR) of BxbI. The CDS was subsequently assembled in a multipartite reaction with the P35S promoter with no ATG, the GFP coding sequence and the T35S terminator.

The plant codon-optimized BxBI gene (BBa_K1742005) was domesticated and standardized as a GoldenBraid part and cloned into the pUPD2 entry vector. The BxbI integrase gene was then assembled in a multipartite reaction with the strong promoter from the Cauliflower Mosaic Virus (P35S), and its terminator (T35S). With a binary GoldenBraid assembly step we obtained a multigenic construct (BBa_K1742009) of the previously described transcriptional units. This construct was then transformed, by agroinfiltration, into N. benthamiana plants for testing the expression of GFP.

Figure 1. Expression levels of GFP in N. benthamiana leaves. A) Agrobacterium-mediated transformation with the multigenic construct BBa_K1742009. B) Plant leaf transformed with the PhiC31 reporter element assembled with the promoter, GFP and the terminator.

References

1. Ghosh, P., Pannunzio, NR., Hatfull, GF., and Gottesman, M. (2005). Synapsis in phage Bxb1 integration: Selection mechanism for the correct pair of recombination sites. Journal of Molecular Biology, 349(2), 331–348.

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
  Illegal NgoMIV site found at 49
  
• 1000
  COMPATIBLE WITH RFC[1000]