Gene Synthesis, Expressing, Recombination Activity Of β Protein, β Protein 177 And FLP

Recombineering is a newly-developed genetic engineering technology in recent years. Based on recombinase and homologous recombination, recombineering technique can simplify the experimental operation and shorten the test period due to reducing the complex steps of enzyme digestion and ligation. Recombinase-mediated integration greatly improves the efficiency, accuracy and adaptability of the recombination. As a reslut, recombineering is recognised as the most important progress and breakthrough in genetic engineering. Recombinase β-protein can be used as a single annealing protein promoting the annealing of DNA complementary strand which improves the efficiency of homologous recombination. Recombinase FLP can identify the an FRT sites and function the sequences for exchange, recombination, deletion and reversion the two orientated FRT sites.They have been successfully applied in the improvement of microorganism. Aiming to improve the traditional methods of microbial breeding, this work focused on gene cloning, expression, purification and the activity analysis of β-protein and FLP, in order to establish a solid foundation for the commercial development and thire application. The results are as follows:(1) The βpro gene encoding P protein desigened according to E. coli-codon-preferred was artificially systhesised by overlapping PCR and the gene was 100%identify with the designed sequencing. The pET30a-βpro was constructed and overexpressed in E. coli BL21(DE3) in optimal express condition. Analysis and detecting by SDS-PAGE, the protein bands of about of 28 kD which expected molecular mass of p-pro was visualized clearly.(2) To improve the stability and soluble expression quantity, the redundant structure of P protein must be removed. So an expression vector of pET30a-βpro177 was constructed and transformed to E. coli BL21(DE3). In the optimized expression condition with IPTG as the inducer, the soluble expression quantity of β protein 177 increased. The result of DNA hyperchromic effects test indicated that P protein with 177 amino acid residues could promote the annealing of the single DNA.(3) An expression vector of pQE32-flp was constructed and overexpressed in E. coli M15 in the optimized express condition. Analysis and detecting by SDS-PAGE, the protein bands of about of 45 kD was accordance with the theoretical molecular weight of FLP. PMD18-FRT-kan-FRT were designed and constructed in order to detect the activity of FLP. Agarose gel electrophoresis results showed that FLP purified could delete the DNA sequence between two homonymous FRT sites Vector pMD18-FRT-kan-FRT as the substrate.(4)PBBlMCS-psldh-flp, pBBlMCS-ptufb-flp and pBBlMCS-pgen-flp which flp was combined with promoter sldh, tufb and gen were constructed and transformed to Gluconobacter suboxydans. The positive transformant was obtained from the above process. The all promoter could effectively start the transcription and expression offlp in the host cell by two-step RT-PCR verification. The result lays a foundation of further study on the genetic modification of Gluconobacter suboxydans.