Part:BBa_K1875012

This part produces a guide RNA that pairs with an operator.

This basic part can be used to express a guide RNA (gRNA) from [http://2016.igem.org/Team:BostonU BostonU 2016’s project Gemini]. Specifically, this part expresses gRNA 8, the 20bp target sequence 5’-GTTGCGCGTCCGTATCAAGG-3’. This sequence can be found in basic part BBa_K1875006 and composite part BBa_K1875015, an operator reporter from the Gemini Library.

gRNA expression vectors were transiently transfected into HEK293FT cells with guide to validate the functionality of this part. These vectors were co-transfected with dCas9-VPR and its paired gRNA operator reporters containing gRNA 8, a mini CMV (BBa_K1875000), a Kozak (BBa_K1875001), a GFP (BBa_K1875003), and a rabbit beta globin poly-A terminator (BBa_K1875002). GFP fluorescence was assayed using flow cytometry to compare expression levels both with and without the gRNA expression vectors. Results indicated that there was low basal expression without gRNA 8 and a high level of expression with gRNA 8. The Gemini system was compared to a CMV from the registry (Part BBa_I712004) to test its strength against a different promoter. The parts containing gRNA 8 from the Gemini system were found to be stronger than the CMV.

gRNA expression vector plasmid map

Screen of GFP expression in gRNA operator reporters with and without gRNA expression vectors

BostonU 2016 Project description

CMV vs Gemini operators

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 247
  
• 1000
  COMPATIBLE WITH RFC[1000]