Part:BBa_K3037002

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

For mutate it the primers used were: Forward: CTTTTCGtATTCCTTATTATGTTGGTC Reverse: CATAATAAGGAATaCGAAAAGTCAAG