Part:BBa_K1875010

Base 10 target mutation to make mutated guide operator 13

Target sequence g13 is expressed by a guide RNA (gRNA) expression vector from the [http://2016.igem.org/Team:BostonU 2016 BostonU] Gemini Library. The gRNA binds to dCas9-VPR and targets a paired gRNA operator reporter to activate a downstream gene of interest (figure 1). One way to decrease expression of the gRNA operator reporters is to mutate the guide target sequence of the guide operator (the guide target sequence followed directly by the PAM). The 2016 BostonU iGEM team made single point mutations on each base of the guide target sequence to test the binding affinity of dCas9. The point mutation was determined by whether the base was a purine or a pyrimidine and the complement of the base. If the base was a purine, it was mutated to be a pyrimidine and of the opposite coupled nucleotide (i.e: A <-->C, G<-->T). Results concluded that the expression dramatically decreased at base 10 of the target sequence (figure 2). Due to its significantly lower expression when placed in their operator reporters, the BostonU team submitted chose to submit BBa_K1875019 which contains gRNA 13. Part BBa_K1875016, an operator reporter with one binding site, also contains the same target sequence.

Figure 1: [http://2016.igem.org/Team:BostonU BostonU 2016] Project description

Figure 2: Screen of GFP expression in an operator reporter (containing g13) with sequentially mutated base.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]