The Method Of Constructing Gene Library Based On Motif Recombination

The generating gene library diversity and constructing a highly efficient screening system are the key steps of directed enzyme evolution. In this paper, we constructed a high efficiency combined expression and screening vector by agaB gene producing clear zones around colonies when expressed on solid LB medium. In addition, we also constructed a diversity gene library by reassembly PCR.1. We used the stable and efficient pBluescript II KS (+) cloning plasmid as the template and inserted the promoter consistent with the direction of Lac promote to construct the constitutive expression vector. The constructed expression vector was screened by using blue-white screening and modified by adding the restrictive endonuclease site of pET-24a(+). AgaB gene was inserted into the resulting expression vector to build a screening vector. AgaB gene can produce clear zones around colonies on solid LB medium when it is expressed. The colonies with no clear zones around them are positive clones when gene fragments were inserted into agaB gene. The colonies with clear zones around them were false-positive clones owing to agaB gene being interrupted. The kanamycin resistance gene was used to test the efficiency of the screening vector. There were 15 positive clones in selected 16 colonies by colony PCR and agarose gel electrophoresis and the screening efficiency was more than 90%, which was higher than the traditional screening vectors. The constructed vector simplified the experimental procedure, reduced the cost of experiments, and not depended on blue-white screening.2. A large-capacity and diversity gene library was constructed. Firstly, we designed two 21 bp random sequences as the basic modules of reassembly PCR. The both ends of random sequences were added 9 bases as general connectors in the reassembly PCR. The selected nine bases could be encoded to form flexible amino acids as linker. The 21 nt random sequences were random encoding 7 amino acids to form a protein motif required. Therefore, the random sequences could form a large-capacity and diversity gene library which coding large number of proteins by reassembly PCR. To control the length of gene in the process of reassembly PCR, terminated sequences were added to obtain the appropriate size of gene fragments. The reaction system and reaction procedures were optimized, and then tested the diversity of the gene library using conventional methods of molecular biology. We used bioinformatics software BioEdit to test the diversity gene library. As a result, we could find that there are large differences in the sequence and the low homology between the segments. The result showed that the diversity of gene library was very good.