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Team:Duke/Project/Results/Part1
From 2013.igem.org
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<div>As plasmids were constructed, they were confirmed by restriction digest and sequencing. </div> | <div>As plasmids were constructed, they were confirmed by restriction digest and sequencing. </div> | ||
| + | |||
| + | #TALEs: | ||
| + | ##All 6 TALEs with mCherry tags were built and confirmed | ||
| + | |||
| + | #CRISPR: | ||
| + | ##6 sgRNAs were built and confirmed. | ||
| + | ##dCas9-mCherry fusion protein was not cloned successfully, despite many attempts at various cloning protocols. We suspect problems with the source plasmid, or possibly effects of the large insert size. We obtained a dCas9 plasmid without an mCherry fusion that is not yet yeast-integrable. | ||
| + | |||
| + | #Reporters: | ||
| + | ##ACT1pr+yEVenus with no enhancer was built and confirmed | ||
| + | ##ACT1pr+enhancer+yEVenus was built and confirmed for 1 and 3 copies of each enhancer sequence | ||
| + | ##ACT1pr+enhancer+yEVenus with 5 copies of enhancer sequence caused more difficulties, because the enhancer insert was too repetitive to be synthesized by standard means. We split the sequence into six small oligonucleotides for PCR amplification, but the product was messy and not useful. We then designed two large oligonucleotides for each sequence and were able to PCR amplify and clone the resulting insert. In this way we obtained and confirmed constructs with 5 enhancers. | ||
| + | ##After initial testing, we decided to build reporters with TEF1 promoters replacing the ACT1 promoter. TEF1pr+enhancer+yEVenus with 1 and 3 copies of the enhancer were built and confirmed. | ||
Revision as of 04:55, 27 September 2013
Results: Library of parts
As plasmids were constructed, they were confirmed by restriction digest and sequencing.
- TALEs:
- All 6 TALEs with mCherry tags were built and confirmed
- CRISPR:
- 6 sgRNAs were built and confirmed.
- dCas9-mCherry fusion protein was not cloned successfully, despite many attempts at various cloning protocols. We suspect problems with the source plasmid, or possibly effects of the large insert size. We obtained a dCas9 plasmid without an mCherry fusion that is not yet yeast-integrable.
- Reporters:
- ACT1pr+yEVenus with no enhancer was built and confirmed
- ACT1pr+enhancer+yEVenus was built and confirmed for 1 and 3 copies of each enhancer sequence
- ACT1pr+enhancer+yEVenus with 5 copies of enhancer sequence caused more difficulties, because the enhancer insert was too repetitive to be synthesized by standard means. We split the sequence into six small oligonucleotides for PCR amplification, but the product was messy and not useful. We then designed two large oligonucleotides for each sequence and were able to PCR amplify and clone the resulting insert. In this way we obtained and confirmed constructs with 5 enhancers.
- After initial testing, we decided to build reporters with TEF1 promoters replacing the ACT1 promoter. TEF1pr+enhancer+yEVenus with 1 and 3 copies of the enhancer were built and confirmed.
