Part:BBa_K2839016

CRISPRi stabilized J23104 promoter

1.Short Description

This part consists of a short chimeric RNA (sgRNA), a CRISPRi stabilized promoter (J23104) and a fluorescent marker (sfGFP). The sgRNA forms a complex with dCas9, a catalytically inactive form of Cas9 and binds to the target promoter region blocking bacterial transcription initiation.

This part was designed by iGEM Thessaloniki, it is RFC10 compatible and together with the dCas9 cassette can be used to achieve stabilized expression of the sfGFP marker, decoupled from gene/plasmid copy number.

2.Biology and Functionality

CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) systems are naturally found in a plethora of prokaryotic species providing resistance to foreign DNA elements. One of the widely used CRISPR systems is the one derived from Streptococcus pyogenes and is based on the cleavage activity of Cas9 which together with two RNA molecules(crRNA and tracrRNA [1] binds and cleaves a specific target site (endonuclease). dCas9 is a catalytically inactive Cas9 nuclease modified with two single point mutations which can still tightly bind at the target DNA but lacks endonuclease activity. CRISPR interference is a DNA editing technique that allows for control in gene expression. It requires the presence of a short chimeric RNA, which forms a complex with the dCas9 protein and determines the binding specificity of the complex at the target DNA.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 194
  Illegal NheI site found at 463
  Illegal NheI site found at 486
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1042
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]