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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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE 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 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2170
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