Week1

Construct t7-lac inducible dCas9,pcyA and Ho1 plasmid

Sequencing result showed both plasmids were accurately constructed.

Amplify ho1 sequence using new primers to add NdeI and XhoI restriction sites.

Digest T7-pcyA-pRSFDUET plasmid and PCR product and then purify.

Ligation and transformation. (3 hours, DH5α). Culturing overnight.

Pick colonies and identify.

No positive results showed because of contaminants.

Construct Red Sensor plasmid.

Considering low copy number of pCDFDuet, we change the constitute promoter into T7 promoter, an inducible one.

PCR from constructed cph8-pSB1C3 plasmid to get cph8 sequence combined with terminator (adding restriction enzyme cutting sites, Nco I and Hind III).

Digest cph8-ter sequence and pCDFDuet with Nco I, 6 hours and Hind III, 1 hour.

Ligation and transformation. (3 hours, DH5α). Culturing overnight.

Plasmid of cph8-pCDFDuet of constitutive promoter is confirmed by sequencing.

Overlap PCR to get sgRNA sequence.

Construct green Sensor plasmid.

Sequence of green sensors on pCDFDuet dis confirmed by sequencing.

Inserted sgRNA is confirmed by PCR and digestion.

Co-transformation: RFP, green sensor with sgRNA, dCas, segment.

Get the Blue sensing part

After getting the blue sensing part, next we will link the sgRNA to it

Week2

Construct Red Sensor plasmid.

Picking colonies of T7 promoter and culturing 12 hours.

Extract the plasmid through miniprep. Identification by PCR, using primers of cph8 and primers on pCDFDuet).

Picking samples with positive results, identification by digestion(Kpn I and Hind III).

Digest cph8-pCDFDuet and sgRNA with Kpn I, 6 hours and Hind III, 1 hour.

Ligation and transformation. (3 hours, DH5α). Culturing overnight.

Picking colonies of constitutive promoter and culturing 12 hours.

Keep Ligating

Strange things keep happening and we have to ligate the sgRNA to it. So harsh.

Week3

Construct Red Sensor plasmid.

Extract the plasmid through miniprep. Identification by PCR, using primers of cph8 and primers of sgRNA).

Picking samples with positive results, identification by digestion(Xba I and Hind III).

No positive results, ligation and transformation again.

Sensor+sgRNA of blue light controlled gene regulating system

Finally we get the right plasmid, next we need to test its function. Ready to set case group and control group.