Coding

Part:BBa_K1742005

Designed by: Ivn Casas Rodrigo   Group: iGEM15_Valencia_UPV   (2015-09-04)

BxBI integrase

Bxb1 is a protein from Mycobateriophage Bxb1’s gp35 gen. Furthermore, a CAT1 intron from Ricinus communis has been added before exon in order to increase the efficiency of the enzyme. 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 Valencia_UPVBxb1_circuit.png

Biology and Usage

The Nicotiana benthamiana codon-optimized BxBI gene 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 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.

800px-ValenciaUPV_bxb1_GFP.png

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. Kim, A.I., Ghosh, P., Aaron, MA., Bibb, LA., Jain, S., and Hatfull, GF. (2003). Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene. Molecular Microbiology, 50(2), 463–473
2. 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
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1556
    Illegal BamHI site found at 738
    Illegal XhoI site found at 257
    Illegal XhoI site found at 743
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1057
    Illegal NgoMIV site found at 1295
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//chassis/eukaryote/nbenthamiana
Parameters
None