Part:BBa_K1875009

Three repeated guide operators to make triple 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 increase expression of the gRNA operator reporters is to have repeated guide operator sequences (the guide target sequence followed directly by the PAM). The 2016 BostonU iGEM team made a triple multimerized guide operator with several spacer sequences between each guide operator to test the binding affinity of dCas9. Results concluded that the highest expression was seen among a 24 bp spacer, the longest spacer sequence they tested (figure 2). Due to its high expression when placed in their operator reporters, the BostonU team submitted chose to submit BBa_K1875018 which contains gRNA 13. Parts BBa_K1875016, an operator reporter with one binding site, and BBa_K1875017, an operator reporter with two binding sites, contain the same target sequence.

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

Figure 2: Screen of GFP expression in double and triple multimerized operator reporters (containing g13) with and without the gRNA expression vector

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]