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.
Point Mutation of Blue Sensor
Luckily we got a right clone from the plate
Sadly it has a point mutation that ends the transcription of our protein
Time to perform point mutation.
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.
Still Point Mutation of Blue Sensor
Due to the low efficiency of the first pair of point mutation primers we designed, we cannot get the right one.
Still doing point mutation
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.
Result of Point Mutation
Finally we get 2 clones, after sequencing we find there are 24 tedious bps, the other, 3 tedious bps.
Sad but maybe the second can be used.
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.
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