Difference between revisions of "Part:BBa K3037002"
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|'''Use in'''
 
|'''Use in'''
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|Escherichia coli
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|<span style="font-style: italic;">Escherichia coli</span>
 
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|'''RFC standard'''
 
|'''RFC standard'''
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The TU Dresden 2019 team design this biobrick in order to make a fusion protein with dCas9 in accordance to the RFC 25 standard. [https://2019.igem.org/Team:TU_Dresden (more information)]
 
The TU Dresden 2019 team design this biobrick in order to make a fusion protein with dCas9 in accordance to the RFC 25 standard. [https://2019.igem.org/Team:TU_Dresden (more information)]
  
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dCas9 was inserted into the pSB1C3 vector for transformation and expressed in E. coli .  
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dCas9 was inserted into the pSB1C3 vector for transformation and expressed in <span style="font-style: italic;">Escherichia coli</span>.  
  
  
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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.  
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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 <span style="font-style: italic;">E. coli</span>.  
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New scope of in vitro applications of Cas9, which is normally used in vivo mainly
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New scope of in vitro applications of Cas9, which is normally used in vivo mainly.
  
  
 
=== Biology ===
 
=== Biology ===
  
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Part of the CRISPR System
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Part of the CRISPR System. Immune system of bacteria, which store sequences of viral infections, recognize them and cut them apart.
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Immune system of bacteria, which store sequences of viral infections, recognize them and cut them apart
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Mutation to not cut
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We can use the system by providing guideRNAs to locate to any target DNA sequence with a high specificity.
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We can use the system by providing guideRNAs to locate to any target
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Implemented in many engineering approaches of mammalian cells
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Has mutation in the Ruv site and therefore it has no endonuclease function, only binding to the DNA.
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But we make it possible to be used by overexpressing it in microbes
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Can bind any sequence of interest given by the guide RNA
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Has mutation in the Ruv site and therefore no endonuclease function (only binding no cutting)
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Revision as of 19:29, 12 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 Escherichia 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.

We can use the system by providing guideRNAs to locate to any target DNA sequence with a high specificity.

Has mutation in the Ruv site and therefore it has no endonuclease function, only binding to the DNA.


Sequence


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1096
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3375
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Design Notes

- Mutated EcoRI site in the midde of the coding region by site directed mutagenesis PCR

References