Construction And Expression Of The Prokaryotic Biobrick Recombinant Expression Vector For The Multi-copy Mannanase Gene

In the fields of biological research, protein expression is a very important content, researchers often pursue a high efficient expression of target protein, in order to achieve the objective of the study. However, there will always encounter some difficulties, the protein is difficult to effectively express, or the expression level could not meet our needs,and a variety of ways had been introduced to solve these problems.Based on the purpose of this,this study seek the way in methodology on higher and more effective expression of the target protein,this will provide a feasible mean for the expression of certain proteins, by increasing the gene copy numbers to increase the yields of the target protein.The biobrick assembly technique, with a fast and efficient assembly,is developed in recent years, The present study take the principle of biobrick, to build multiple copies of the prokaryotic expression biobrick vector of the mannanase gene (pBAD/His-man) quickly, and then express in the bacteria E.coli BL21(DE3),comparing the protein expression levels among various genes copies in the same external conditions, and then analyse the differences, to reveal the connection between the protein expression level and the gene copies. Endogenous regulation of prokaryotic gene expression could be complicated, so this study experimented with different inducer concentration and induction time.This will be a good way to eliminate the deviation of the experimental results to some content.This experiment constructed1-6copies mannanase prokaryotic expression biobrick vector pBAD/His-man successfully, after repeated experiments in this study.The results showed that,when the induction time is4hour,the average expression of the2-6copies transformants has no significant difference,but has the higher expression compared to1copy. If we shorten the time to30minutes, the expression level of each copy transformants would exhibit a notable difference, however it’s not certain that which copy transformants has the highest expression,mainly because the expression is not steady. Overall,the increase of the gene number can improve protein expression to some extent indeed.