Part:BBa_K1875018

This operator, when paired with a guide RNA, expresses GFP.

Triple Operator

The BostonU 2016 iGEM team’s Gemini Library contains analog parts in addition to their digital parts. They synthesized multimerized binding sites to increase the expression of the operator. This hypothesis was supported by analogous results in previous synthetic transcriptional regulator research. A multimerized operator means that there are consecutive operators upstream of the promoter where the dCas9-VPR and guide RNA will bind to. In addition to multimerizing operator sites, BostonU 2016 also modulated the space between the binding sites. Figure 2 demonstrates a screen with this multimerized operator containing the target for g13 from the Gemini Library (guide sequence: GTTCTAAACGTTGGTCCGTC). BostonU 2016 chose to submit the strongest of its triple multimerized operators which had a 24 base pair spacer between the operators.

Figure 1: Triple multimerized gRNA operator reporter plasmid map

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1021
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]