Week1

Construct Red Sensor plasmid.

Plasmid is confirmed by sequencing.

Transform pCDFDuet plasmid to DH5αin large scales for 24 hours.

Extract the plasmid through miniprep.

PCR to get cph8 sequence combined with promoter and terminator(adding restriction enzyme cutting sites, Xba I and Hind III).

Digest pro-cph8-ter sequence and pCDFDuet with Xba I and Hind III, 6 hours. Purification pCDFDuet with gel extraction.

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

Week2

Construct Red Sensor plasmid.=

Picking colonies and culturing 24 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(Xba I and Hind III).

Sequencing results shows that promoter and terminator have been misconnected. Intended to shorten digestion time of Hind III.

Week3

Construct Red Sensor plasmid.

PCR to get cph8 sequence combined with promoter and terminator.

Digest pro-cph8-ter sequence and pCDFDuet with Xba I, 6 hours and Hind III, 1 hours. Purification pCDFDuet with gel extraction.

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

Week4

Construct Red Sensor plasmid.

Picking colonies and culturing 24 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(Xba I and Hind III).

We found that when plasmids duplicate in pCDFDuet, they could get higher concentration and purity only on longer than 12 hours. Intend to shorten culturing time.

Construct green Sensor plasmid.

Insert sgRNA which points to RFP in downstream of Ccas in opposite direction.