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Team:Hawaii/Construction of Omega Interposon BioBrick
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* Cut pSMC121 with SmaI or XmaI; cut pSB1A3 with the same enzyme. | * Cut pSMC121 with SmaI or XmaI; cut pSB1A3 with the same enzyme. | ||
| - | * Purify | + | * Purify restriction digests to get rid of small digest fragments. |
* Ligate the omega interposon with pSB1A3 | * Ligate the omega interposon with pSB1A3 | ||
* Transformation: | * Transformation: | ||
| - | # Transform DH5-a with the ligated product | + | # Transform DH5-a with the ligated product, select on Amp100. |
| - | # Transform DB3.1 | + | # Transform DB3.1(ccdB resistant strain) with the ligated product, select on Amp100 as a negative control. If the experimental plate has no colonies and the (-) control does, this means that the intramolecular ligation of pSB1A3 was the favored reaction. |
| - | + | ||
== Results == | == Results == | ||
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== Discussion == | == Discussion == | ||
| - | * | + | * All components of this experiment will be cut with the same restriction enzyme. This means there will probably be a large background of intramolecular ligations (pSB1A3 will ligate back to itself and pSMC121(minus the omega interposon)will ligate to itself as well). The negative control should let us know the degree to which this will occur. |
{{Team:Hawaii/Footer}} | {{Team:Hawaii/Footer}} | ||
Revision as of 23:52, 9 August 2008
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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 restriction digests to get rid of small digest fragments.
- Ligate the omega interposon with pSB1A3
- Transformation:
- Transform DH5-a with the ligated product, select on Amp100.
- Transform DB3.1(ccdB resistant strain) with the ligated product, select on Amp100 as a negative control. If the experimental plate has no colonies and the (-) control does, this means that the intramolecular ligation of pSB1A3 was the favored reaction.
Results
Discussion
- All components of this experiment will be cut with the same restriction enzyme. This means there will probably be a large background of intramolecular ligations (pSB1A3 will ligate back to itself and pSMC121(minus the omega interposon)will ligate to itself as well). The negative control should let us know the degree to which this will occur.
