Part:BBa_K3921004

oriC4

OriC4 refers to No.4 oriC binding site tested by Wiktor’s group [1] which is proven to be functional of inhibiting replication initiation. When oriC is inhabited, E.coli cells can trigger neither replication initiation nor cell division.

We named our sgRNA that binds No.4 oriC binding site as “oriC4”. To test it, we used an old version of dCas9 plasmid where dCas9 was expressed by J23103, coupled with a CcaS-CcaR plasmid where oriC4 was expressed by PcpcG2-68 promoter (plasmids were provided by our host lab). These two plasmids were co-transformed into DH5a strain. Light-control experiment was performed in a 24-well plate adapted device. Overnight culture was diluted to 50% and then shaken at 37°C, 200rpm, for 2 hours under green light. The bacteria suspensions became clarified with cells deposited at the bottom and became floccus after shaking. This phenomenon indicates the expression of oriC4 forced cells to become filamentous and possibly died during shaking.

Reference:
[1]. Wiktor J, Lesterlin C, Sherratt DJ, Dekker C. CRISPR-mediated control of the bacterial initiation of replication. Nucleic Acids Res. 2016 May 5;44(8):3801-10.

Usage and Biology

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]