Part:BBa_K1742009

35S:BxbI:T35S - 35S:ReporterBxBI::GFP:T35S

Bxb1 is a protein from Mycobateriophage Bxb1’s gp35 gen. Its function is to regulate the lysogenic cycle of the phage by integrating and excising phage’s genome in Mycobacterium smegmatis chromosome [1]. 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”)[2]. Depending on the position and sense of these sites, bxb1 is able of excising or inverting. We used bxb1 as excisionase by flanking a sequence close to the promoter

Biology and Usage

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).

In order to see the activity of the integrase, Valencia_UPV 2015 designed a 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. With a binary GoldenBraid assembly step we obtained the present 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.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 1959
  Illegal BamHI site found at 1141
  Illegal XhoI site found at 660
  Illegal XhoI site found at 1146
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 4
  Illegal NgoMIV site found at 49
  Illegal NgoMIV site found at 1460
  Illegal NgoMIV site found at 1698
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2170