Stage one：basal experiments

Introduction

As the construction and concentration of recombinant plasmid rely heavily on E.coli system in current molecule experiment protocols of Bacillus subtilis, Bacillus subtilis acts only as the secretory expression vector. Therefore, to verify the practicality of our locus and the transdermal function of recombinant transdermal protein on top of its original functions, we conducted basic experiments on E.coli to verify our assumptions.

The following figure shows the stages of our basal experiments:

Results

① E.coli BL21 proved that the standard transdermal locus does work with GFP.

Using GFP to prove the validity of a newly designed circuit is a classical way to verify the expressing of this circuit. As expressions in E.coli involve neither secretory nor sequential problems, we hoped to verify the practicality of our locus by the expression of TD1-GFP. Thus we selected pET22b,which is a common recombinant vector for plasmid construction, as our recombinant vector and E.coli BL21 as engineered bacteria . We fused sequence TD1-GFP with T7 promoter from pET22b downstream and succeeded in expressing fusion protein TD1-GFP, induced by IPTG.

The molecule weight and key marker data of fusion protein TD1-GFP