Part:BBa_K578001
E. coli CRISPR I leader sequence
Usage and Biology
- This is the AT-rich leader region of the CRISPR I array in Escherichia coli str. K-12 substr. MG1655. "Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS" (Ü. Pul et al, 2010) characterized a single promoter (Pcrispr1) in this sequence. Transcription is silenced through H-NS binding to the leader sequence. Induction requires anti-silencing, either by protein-independent processes, DNA-binding proteins or other modulatory mechanisms not yet identified.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Confirmation of Part Length
PCR of BBa_K578001 using Primers VF2 (BBa_G00100) and VFR (BBa_G00101) from pSB1C3. The part is 106bp plus 42bp for the Standard BioBrick prefix and suffix. Note: The primers add approximately 200bp to the BioBrick's size (The primary band visualized is larger than expected an requires sequencing confirmation).
Confirmation of Function
ASU iGEM 2011 has tested this part by designing an experiment that utilizes the endogenous Cas genes of E. coli K-12 MG1655. As expected, a very clear difference is seen when comparing the transformation efficiency (denoted in the graph by weighted colony count) of bacteria containing Cas genes and a Leader+RSR construct, and bacteria containing only Cas genes and a Leader.
This indicates that, as designed, this K12 leader sequence promotes the succeeding RSR array, producing a clear and consistent difference in weighted colony count. This also indicates that the interaction is also with the Cas genes of the K-12 MG1655, as the mirrored combination of transformations in E. coli BL21 DE3 (which does not have endogenous Cas genes) does not show this difference.
More information on the experimental protocol can be found on our [http://2011.igem.org/Team:Arizona_State/Project/E_coli wiki].
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