Difference between revisions of "Part:BBa K3037002"
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There are many dCas9 biobricks already available but all of them are optimized for expression in mammalian cells. This is the first one that is codon optimized to be expressed in E.coli. | There are many dCas9 biobricks already available but all of them are optimized for expression in mammalian cells. This is the first one that is codon optimized to be expressed in E.coli. | ||
New scope of in vitro applications of Cas9, which is normally used in vivo mainly | New scope of in vitro applications of Cas9, which is normally used in vivo mainly | ||
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Revision as of 17:39, 6 October 2019
dead CRISPR Associated Protein (dCas9)
| dCas9 | |
|---|---|
| Function | Expression |
| Use in | Escherichia coli |
| RFC standard | RFC 25 compatible |
| Backbone | pSB1C3 |
| Submitted by | Team:TU_Dresden 2019[1] |
Overview
The TU Dresden 2019 team design this biobrick in order to make a fusion protein with dCas9 in accordance to the RFC 25 standard. (more information)
dCas9 was inserted into the pSB1C3 vector for transformation and expressed in E. coli .
There are many dCas9 biobricks already available but all of them are optimized for expression in mammalian cells. This is the first one that is codon optimized to be expressed in E.coli.
New scope of in vitro applications of Cas9, which is normally used in vivo mainly
Biology
Part of the CRISPR System Immune system of bacteria, which store sequences of viral infections, recognize them and cut them apart Mutation to not cut We can use the system by providing guideRNAs to locate to any target Implemented in many engineering approaches of mammalian cells But we make it possible to be used by overexpressing it in microbes Can bind any sequence of interest given by the guide RNA Has mutation in the Ruv site and therefore no endonuclease function (only binding no cutting)
Sequence
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1096
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3375
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
- Mutated EcoRI site in the midde of the coding region by site directed mutagenesis PCR