Boston UniversityPlasmidChoice
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== Plasmid Choice== | == Plasmid Choice== | ||
| - | Our plan is to replicate our engineered plasmid in E. coli and then transfer the plasmid to S. oneidensis through conjugation. | + | Our plan is to replicate our engineered plasmid in E. coli and then transfer the plasmid to S. oneidensis through conjugation. In choosing our plasmid there were several points we had to consider, they are as follows: |
| + | |||
| + | # The plasmid CAN be properly replicated in S. oneidensis and will persist in future generations of transformed bacteria. This can be ensured by the presence of the origin of replication p15A. | ||
| + | # The plasmid contains origins of transcription oriT to allow conjugal transfer from E. coli to S. oneidensis, and preferably (though not necessarily) oriV for vegetative replication of the plasmid. | ||
| + | # A minimal number of other major genes in order to avoid unexpected, confounding effects when attempting to evaluate the efficacy of directed evolution in targeted genes. | ||
| + | # A robust number of cleavage sites which will allow for the transposing of two genes into the plasmid. | ||
| + | |||
| + | We ended up selecting plasmid pJQ200 as it matches the above criteria for the most part. It contains origins of transcription p15A, oriT, and oriV. There are three major gene sites, gtmR (gentamycin resistance), sacB (lethal sucrose sensitivity), and traJ (related to conjugation, but is redundant when the chromosomal DNA of the E. coli strain we are using is taken into account--thus non-essential). We are planning on using restriction enzymes to transpose two additional genes into the pJQ200, one each into the sacB and gtmR sites. The reasons for this decision will be detailed in a further update. | ||
| + | |||
This is a [https://www.atcc.org/common/images/vectors/gifs/77482.gif map] of PJQ200, not engineered. | This is a [https://www.atcc.org/common/images/vectors/gifs/77482.gif map] of PJQ200, not engineered. | ||
Revision as of 18:07, 8 June 2007
Plasmid Choice
Our plan is to replicate our engineered plasmid in E. coli and then transfer the plasmid to S. oneidensis through conjugation. In choosing our plasmid there were several points we had to consider, they are as follows:
- The plasmid CAN be properly replicated in S. oneidensis and will persist in future generations of transformed bacteria. This can be ensured by the presence of the origin of replication p15A.
- The plasmid contains origins of transcription oriT to allow conjugal transfer from E. coli to S. oneidensis, and preferably (though not necessarily) oriV for vegetative replication of the plasmid.
- A minimal number of other major genes in order to avoid unexpected, confounding effects when attempting to evaluate the efficacy of directed evolution in targeted genes.
- A robust number of cleavage sites which will allow for the transposing of two genes into the plasmid.
We ended up selecting plasmid pJQ200 as it matches the above criteria for the most part. It contains origins of transcription p15A, oriT, and oriV. There are three major gene sites, gtmR (gentamycin resistance), sacB (lethal sucrose sensitivity), and traJ (related to conjugation, but is redundant when the chromosomal DNA of the E. coli strain we are using is taken into account--thus non-essential). We are planning on using restriction enzymes to transpose two additional genes into the pJQ200, one each into the sacB and gtmR sites. The reasons for this decision will be detailed in a further update.
This is a map of PJQ200, not engineered.
