We have successfully ligate tandem double promoters with J23102 J23106 and J23116 in different orders: J23102-J23102, J23102-J23106, J23106-J23102, J23116-J23102, J23106-J23106, J23106-J23116, J23116-J23106. And we have submitted these new biobrick. Unfortunately, other combination failed because of mutation showed by sequence result.
And we have detected the transcription strength of these tandem double promoters.
Guide RNA
We chose tandem double promoters J23106-J23116 as our target. Based on this, we designed serval targeting sites, including both activation(A1,A2,A3,A4,A5) and repression(R1, R2) sites after searching the PAM region. And through a three-cycle overlap PCR, we construct these functinal guide RNA.
Here shows the agarose electrophoresis:
dCas9 Devices
First, we add dCas9 gene( amplify through PCR) to express vector with promoter to construct a new biobrick. We name this new biobrick BBa_K1081000.
Here shows the agarose electrophoresis and SDS-PAGE of BBa_1081000:
Then we ligate omega subunit gene downstream of dCas9.
Here shows the agarose electrophoresis of the product:
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