Team:SHSBNU China/Improve
Improve
Overview
Previously, there was only CsgA part uploaded in iGEM parts website. Our Team improved part Part BBa_K1583000 by adding a SpyTag sequence. It fused to gene csgA, enabling CotA laccase to be fixed onto the biofilm to form a covalent bond--SpyTag-SpyCatcher.
Improvement Details
By using sfGFP-SpyCatcher protein, we tested the binding efficiency of the covalence SpyTag-SpyCatcher since sfGFP is a non-toxic and common used flurorescent protein. Gene csgA on the plasmid of pET28a was transferred into ΔMG1655 as control group. Gene csgA – spytag on the plasmid of pET28a was transferred into ΔMG1655 as experiment. The experiment was divided into 4 groups. The bateria in the Reaction Stock had neither csgA nor SpyTag in neither genome nor plasmid; the ones in Group 1 had csgA in the genome; the ones in Group 2 had sfGFP in the plasmid; the ones in Group 3 had csgA-SpyTag in the genome and the plasmid. The treatment of the experiment is followed by the procedure below:
The liquid after centrifugation was extracted and measured fluorescence.
As can be seen from the result, the experiment group showed the most decrease of sfGFP-SpyCatcher protein. The difference between the control group and experiment group was significant. Thus we could confirm our CsgA-SpyTag system was functional.