Week1

Template Plasmid Amplification from Kit Plate

We amplified BBa_K592006, BBa_K592016, BBa_K592018, BBa_J23100, BBa_B0015, BBa_I15009, BBa_I15008 and BBa_J23119 from kit plate for further construction.

Construct consititutional backbone plasmid

PCR to add SpeⅠand Sal I restriction sites upstream of and Pst I downstream of BBa_B0015 terminator part. Use spe I and Pst I to digest the PCR product and the BBa_K880005（promotor and rbs) part and then use T4 ligase to connect PCR product and backbone. Identify by monoclonol colony PCR to find positive clone. Sequencing results showed accurate construction of the consititutional backbone plasmid.

Construct luciferase plasmid.

PCR to connect luciferase into pETDuet. (Adding restriction enzyme cutting sites at both ends, Nco I and Xho I)

Recombination of luciferase (Nco I and Xho I)and pETDuet (Nco I and Xho I). Digestion 6 hours and ligation 3 hours.

Transform constructed plasmid to competent cell DH5α. Culturing overnight.

Week2

Construct luciferase plasmid.

Picking colonies and culturing overnight.

Extract the plasmid through miniprep. Identification by PCR.

Construct dCas9, pcyA and Ho1 plasmid

PCR to connect dCas9, pcyA and Ho1 into pSB1C3. (Adding restriction enzyme cutting sites at both ends,Xba I and Sal I)

Digest with Xba I and Sal I, and pSB1C3 backbone with Spe I and Sal I. 6 hours.

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

Picking colonies and culturing overnight.

Identify by monocolonal colony PCR and send 2 copies of positive colony each of Ho1 and PcyA.

No positive dCas9 colony identified.

Construct Red Sensor plasmid.

PCR to connect cph8 into pSB1C3. (Adding restriction enzyme cutting sites at both ends,Xba I and Sal I)

Digest cph8 with Xba I and Sal I, and pSB1C3 with Spe I and Sal I. 6 hours.

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

Picking colonies and culturing overnight.

Construct Blue Sensor plasmid

Add suitable enzyme cutting site though PCR

Digest and ligate, to get the constitutive operon of blue light sensor.

Week3

Construct luciferase plasmid.

Plasmid is confirmed by sequencing.

Construct dCas9, pcyA and Ho1 plasmid

Sequencing reseult showed that the plasmid we got was the model plasmid. The new backbone and original sequence model are both chlorampenicol resistant.

Reconstruct constitutive pcyA and Ho1 plasmid using DpnI to digest plasmid model.

Get 2 plasmids of pcyA to send sequencing.

Again didn't get positive colony of dCas9 and Ho1.

Construct Red Sensor plasmid.

Plasmid extraction and identification of Cph8-PSB1C3. Identification by PCR, using primers of cph8 and standard primer on pSB1C3, VR&VF2).

Concentration of plasmid is low for sequencing. Culture colonies with positive results in large scale for 24 hours. Plasmid extraction and identification by sequencing.

Construct Blue Sensor plasmid onto another Backbone

After we get the right sequencing result, we begin to use a pair of new primers doing PCR to add a new restriction enzyme site on the PCR pieces.

Cut and Paste, trying to get another plasmid.

Week4

Construct dCas9, pcyA and Ho1 plasmid

Constitutive pcyA-pRSF plasmid construction

PCR to add restriction enzyme cutting sites at both ends of consititutive pcyA operon, Xho1 and BamH1)

Digest pRSF-duet1 plasmid and PCR product with Xho1 and BamH1. Use elecrophoresis and gel extraction to purify pcr product and backbone sequence.

Ligation and transformation (3 hours, DH5α).

Picking colonies and culturing overnight.

Identify by monocolonal colony pcr.

Constitutive dCas9 and Ho1 pSB1C3 plasmid construction

Reconstruct Constitutive dCas9 and Ho1 pSB1C3 plasmid.

Got 1 positive colony of Ho1 and sent sequencing.

Again didn't get positive clone of dCas9.

Construct Red Sensor plasmid.

PCR to connect cph8 into pSB1C3.

Digest cph8 with Xba I and Sal I, and pSB1C3 with Spe I and Sal I. 6 hours. Purification pSB1C3 by gel extraction.

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

Picking colonies and culturing 24 hours.

Blue Sensor Plasmid Faces with Challenge

After several tests the result hasn't been got by us.

Repeat and try to find the smallest mistakes.