Team:Heidelberg/Delftibactin/DelRest

From 2013.igem.org

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By the end of last week we decided that the short primers we had ordered in the previous week did not improve amplification of the missing genes from <i>D. Acidovorans </i>. Also the amplification of DelF-G did not proceed as expected. Additionly, further analysis of the Delftibactin cluster, led to the discovery of another putative promotor in front of DelO-P. Therefore we not only decided to design new primers for the region of DelFG but also modified the entire strategy and our construct in concerns of DelOP. To ensure the expression of DelOP in our target organism <i> E.coli </i> we decided to introduce this promotor in our final vector. Logically new primers were ordered for this region but as we still want to use Gibson cloning also the very last fragment of DelA-G has now to be amplified with an overlap, matching the promotor in front of DelOP. The correlating primers can be found in our new vector map.
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By the end of last week we decided that the short primers we had ordered in the previous week did not improve amplification of the missing genes from <i>D. Acidovorans </i>. Also the amplification of DelF-G did not proceed as expected. Additionly, further analysis of the Delftibactin cluster, led to the discovery of another putative promotor in front of DelO-P. Therefore we not only decided to design new primers for the sequenceregion of DelFG but also modified the entire strategy and our construct in concerns of DelOP. To ensure the expression of DelOP in our target organism <i> E.coli </i> we decided to introduce this promotor in our final vector. Logically new primers were ordered for this region but as we still want to use Gibson cloning also the very last fragment of DelA-G has now to be amplified with an overlap, matching the promotor in front of DelOP. The correlating primers can be found in our new vector map.
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                                   <h1>Week 13</h1>
                                   <h1>Week 13</h1>
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In the previous week several of the new primers improved our PCRs for DelO-P. Nevertheless results were not convincing enough to use the amplified fragments for Gibson Assembly. The same encounts for the amplification of DelF-G. Here various strategies where conducted. Though none of these led to an entirely satisfying ampl of the genes DelF and DelG, we were able to amplify regions of the Delftibactin cluster which we could not amplifý with the initial primers. Therefore this week will be used for optimization of these PCRs as well as for validation of the amplicons we were already able to obtain in well established amplifications. Validation will be carried out with various restriction digests. PCR products showing positive digests will be prepared for single read sequencing to validate constructs before Gibson Assembly.
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In the previous week several of the new primers slightly improved our PCRs for DelO-P. Nevertheless results were not convincing enough to use the amplified fragments for Gibson Assembly. The same encounted for the amplification of DelF-G. Here various strategies where conducted. Though none of these led to an entirely satisfying ampl of the genes DelF and DelG, we were able to amplify regions of the Delftibactin cluster which we could not amplifý with the initial primers. Therefore this week will be used for optimization of these PCRs as well as for validation of the amplicons we were already able to obtain in well established amplifications. Validation will be carried out with various restriction digests. PCR products showing positive digests will be prepared for single read sequencing to validate constructs before Gibson Assembly.
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Revision as of 19:42, 2 October 2013

Del Rest. Creating a 32 kbp plasmid.

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Methods: