Week1

Construct dCas9, PcyA and Ho1 plasmid.

PcyA-pRSF plasmid construction

Sequencing results showed both 2 plasmids had been successfully constructed

Ho1-pSB1C3 plasmid construction

Sequencing results showed frameshift mutation in the sequence.

Reconstruct and got 2 extra positive clones and sent sequencing.

dCas9-pSB1C3 plasmid construction

Reconstruct and got 1 positive clone and sent sequencing

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 dCas9, PcyA and Ho1 plasmid

individual dCas9-pRSF plasmid construction

Sequencing result showed accurate construction of dCas9-pSB1C3 plasmid.

Add 2 digestion sites of XbaI and XhoI outside dCas9 operon sequence by PCR.

Digest pRSF-duet1 plasmid and PCR product with XbaI and XhoI.

Ligation and transformation.

No positive colonies identified.

Constitutive ho1-pSB1C3 plasmid construction

sequencing results showed mutation in ho1 operon.

Contacted the ho1 part designer and knew that this protein has some kind of toxicity, which may influence the survival of E.coli.

Considering that Ho1 is the first enzyme in phycocyanobillin production and may product some toxic intermediates.So we decided to connect PcyA and Ho1 first on pRSF-duet1 and then use point mutation.

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 dCas9 ,pcyA and ho1 plasmid

pcyA and ho1 ligation

No positive clone identified .Concerning that the protein may be really toxic, we decided to construct T7-lac inducible ho1 plasmid.

Constitutive dCas9-pRSF plasmid construction.

Reconstruct dCas9-pRSF plasmid and got 2 positive clones and sent sequencing.

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 dCas9， pcyA and Ho1 plasmid

Constitutive dCas9-pRSF plasmid construction

Sequencing results showed dCas9-pRSF plasmid accurately constructed.

This plasmid can be used in blue light system.

T7-lac inducible pcyA, Ho1 and dCas9 plasmid construction

Using new primers to amplify PcyA sequencing （adding BamHI and PstI restriction sites ）

Digest pRSF-duet1 and PCR product and purify.

Ligasing the plasmid and pcr product and transduct.

Picking colonies and identify with PCR.

Send 2 positive clones to get sequenced.

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.

Meanwhile Constructing a T7 clone

Lots of efforts have been put into the constitutively expressed genes and maybe cloning a T7 one is easier.

PCR, cut and paste