Part:BBa_K2483004

regulated dCas9 with sgRNAs and IAA enzymes fused to MS2 and PP7

This is the final part of our metabolic channelling project. Metabolic channelling occurs when enzymes are put in close proximity to each other to decrease diffusion time and increase enzymatic output.

This is the low-copy plasmid of our project, also called the "workhorse". Here, everything is synthesized for the scaffold which is placed on a different plasmid (containing many repeats of either part BBa_K2483005: https://parts.igem.org/Part:BBa_K2483005 or BBa_K2483006: https://parts.igem.org/Part:BBa_K2483006).

This part is designed to work like in the picture below. dCas9 (BBa_K2483002: https://parts.igem.org/Part:BBa_K2483002) binds via the sgRNAs to the scaffold DNA. The sgRNAs have Aptamers attached to them (from iGEM Warwick 2016, BBa_K1994017: https://parts.igem.org/Part:BBa_K1994017 and BBa_K1994016: https://parts.igem.org/Part:BBa_K1994016 were used but the recognition sequence was changed). Those aptamers are designed to bind either MS2 or PP7 (RNA-binding proteins). Additionally, we fused the CDS of IaaM to MS2 and IaaH to PP7 (BBa_K2483000: https://parts.igem.org/Part:BBa_K2483000) so that the fusion proteins bind to the sgRNA and produce Indoleacetic Acid (IAA, Auxin).

It is important to remember that dCas9 is Lac induced because of possible toxicity and growth constraints.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 3004
  Illegal NheI site found at 9656
  Illegal NheI site found at 9930
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4412
  Illegal BglII site found at 6221
  Illegal BglII site found at 9644
  Illegal BglII site found at 9918
  Illegal BamHI site found at 725
  Illegal BamHI site found at 7166
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 5928
  Illegal NgoMIV site found at 8872
  
• 1000
  COMPATIBLE WITH RFC[1000]