Difference between revisions of "Team:Birkbeck/Basic Parts"
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Revision as of 17:36, 8 September 2015
Our BioBricks
Basic Parts
ORF314 (BBa_K1846000)
In the commonly used lab strain of λ-bacteriophage (known as λPaPa), ORF314 is one of two open reading frames resulting from a frameshift in the tail fibre protein gene (stf). This ORF codes for the C-terminus of the protein, which contains the host receptor recognition site. ORF314 binds to the OmpC protein of E.coli. Furthermore, the sequence shows great (>50%) homology with the gene 37 tail fibre protein of bacteriophage T4.
To transform this sequence into a BioBrick basic part, we had the coding sequence synthesised, including the BioBrick prefix and suffix and an additional section to facilitate the cloning of complementary sequence ORF401. We then restricted both our synthesised gene and the linearised plasmid backbones (pSB1C3 for shipping and pSB1K3 for further processing) with EcoRI and PstI, before ligating the two pieces together using T4 DNA ligase. Success of the cloning procedure was confirmed by restriction with EcoRI and PstI followed by DNA agarose electrophoresis (Fig. 1), with pSB1C3 alone and ORF314 alone as controls. The ORF314 biobrick has been assigned BBa_K1846000.
Fig 1. Agarose gel electrophoresis of ORF314 inserted in pSB1C3 and restricted with EcoRI and PstI. The expected band sizes are 1 kb for ORF314 and 2 kb for pSB1C3. Successful results were obtained for samples 2 and 3.