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 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 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 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.