Part:BBa_K2483005

sgRNA target site couples facing each other with 6 bp spacer

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 562
  
• 1000
  COMPATIBLE WITH RFC[1000]

This part is designed to act as a scaffold for dCas9 proteins. Several proteins may bind to one "target cassette" - as we call it. We used this part in our project to recreate metabolic channelling synthetically. Metabolic channelling occurs, when enzymes are put in close proximity to each other, whereby diffusion time is reduced and enzymatic output is increased.

The sgRNA target sites are facing each other which means that one recognition sequence is on the opposite strand of the other and the PAM sequences for the dCas9 are "between" both recognition sequences. As the dCas9 mainly binds in the PAM region, when fusing proteins to dCas9 (or as in our project: via Aptamers to the 3'-end of the sgRNA) the enzymes should be close together and metabolic channelling should occur (see picture).

We inserted a 127 bp "big spacer" between the target site couples to reduce homologous recombination. Nervetheless HDR was a huge problem for us in utilizing this part and PCR and transformation had relatively low efficiency. We greatly encourage to always use different annealing temperatures and template concentration when PCR amplifying this part as most of the time there will be several smaller bands instead of the big one!! We had the best amplification results when working with relatively low template mass (0,1 ng) and annealing temperatures a lot lower than what we got from online Tm calculators (60°C instead of 68°C) but this could just be because of the primers we were using. Additionally, we greatly encourage using E.coli strains which are less susceptible to repeats, for example JM109.

This version of the target cassette has a 6bp spacer between the PAM sequences while the part BBa_K2483006 has 18 bp spacer. There was a version with 12 bp but we could not manage to amplify and transform it.