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Team:Hawaii/Construction of Omega Interposon BioBrick
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Contents |
Construction of Omega Interposon BioBrick
- The omega interposon (OI) BioBrick will be constructed using insertional mutagenesis methods. OI will be inserted into the ccdB region of pSB1A3 by restricting both pSB1A3 (containing ccdB) and pSMC121-a plasmid containing OI- with either XmaI or SmaI, depending on which enzyme is more available.
- The omega interposon is an insertional mutagenesis tool containing the aadA gene from plasmid R1001.1 which confers Spectinomycin and Streptomycin resistance. Flanking the aadA gene are transcriptional termination sites of the T4 gene 32 so that transcription cannot be achieved through the omega interposon from either side. To avoid polypeptide synthesis at the position of the omega interposon, synthetic translational stop codons were also included. Flanking this feature are two polylinkers (Prentki 1983). OI can be used in experiments where disruption of a gene is needed. These experiments are often performed when discerning the function of a gene.
Methods
- Cut pSMC121 with SmaI or XmaI; cut pSB1A3 with the same enzyme.
- Purify the omega interposon from pSMC121 (?) with gel purification.
- Ligate the omega interposon with pSB1A3
- Transformation:
- Transform DH5-a with the ligated product
- Transform DB3.1 cells with ligated product (+) control
- Transform DH5-a with non-restricted pSB1A3 (-) control
Results
Discussion
- This experiment is still under construction
[http://manoa.hawaii.edu/ 
][http://manoa.hawaii.edu/ovcrge/ 
][http://www.ctahr.hawaii.edu 
]
