Team:RDFZ-China/Design
Design
In order to achieve our goals, experiments were conducted in Tsinghua University. To attain quantitative data and manipulate yield of the expression of coding sequence, inducible promoter is being used. K180000 is a ptac promoter consisting LacI coding sequence and preceding ptac sequence. Inducer IPTG can bind LacI to form IPTG-LacI complex, which inhibits the repress from LacI molecule to ptac; thus, downstream coding sequence can be expressed responding to different concentrations of IPTG.
As we noticed that there is no promoter for coding of LacI, we introduced a constitutive promoter K2937000 with RBS to the expression system. This sequence is fused with primers and borne, with reporter GFP, with splicing , and with overlap extension; it then connected with backbone and with Gibson assembly--an in-fusion technique. The plasmid is used for characterization of the composite promoter and for cell free system protein expression.
The region of promoter is consisting of LacI ,constitutive promoter, ptac promoter, and RBS. As said before, we design the prior one to be reverse, and the latter one to be forward. Reasons of doing so are to:
1, Decrease the tendency of such region of leaking
2, Standardize LacI production for future teams for measurement
Therefore, coding sequence can be added directly behind the ptac promoter, and can be expressed by the induction of IPTG. The advantage of the part being used is that there is no more extra characterizes needed besides coding sequence, its tag is used for SDS-PAGE electrophoresis in subsequent steps.
For cell free system protein expression, GFP is preceded with constitutive promoter T7 and constructed on pseva321 backbone.
（See more on results）
