Studies On Screening Of Prokaryotic Enhancer-like Sequences Based On T7 Promoter And Their Functional Regions

Enhancers are an important cis-function element that contain binding sites for transcription factors, and they activate their target genes, but their molecular mechanis of function have not been fully characterized. It is found that the enhancer-liker elements is existed in the genomic DNA sequnce of prokarytic cells and some viruses. Studies have demonstrated that prokaryotic enhancer-like elements are used as a tool to improve prokaryotic expression level. T7 promoter expreesion system is the first choice of prokaryotic expression because it is available for expression of small molecular weight genes but not larger, and there are few research on increasing the expression of high molecular weight foreign genes. Screening new prokaryotic enhancer-like sequenses not only has practical valus to increase the foreign gene expression in prokaryotic cells, but also provides a foundation for its theory of regulation mechnisms.The work includes 4 parts:(1) Construction of enhancer-probing vectors; (2) Isolation of enhancer-like sequence-containing strains and identification of enhancer-like sequence; (3) Analysis of reporter gene expression in enhancer-like sequence-containing strains and Enhancer-like sequences assay; (4) Analysis of the functional region of Enhancer-like sequences.(1) Construction of enhancer-probing vectorsIn this study, major later capsid protein gene L1 (or L11 sequence of truncated L1 gene) of Human Papilloma Virus (HPV) was linked to a reporter gene CAT (chloramphenicol acetyl transfer, CAT). The different molecular weight of two fusion reporter genes were inserted into enhancer-probing vectors. Plasmids were extracted from L1-pMD18-T/DH5? and CAT-pET21a/DH5? strains and constructed the L1-CAT-pET21a enhancer-probing vector by restriction enzyme ligation method. Then, we amplificated L11 gene by PCR, and it was inserted into CAT-pET21a plasmid to construct another L11-CAT-pET21a enhancer-probing vector. The two correct enhancer-probing vectors were confirmed by restriction enzyme digestion and sequencing.Firstly, the functional tests of enhancer-probing strains were carrided out before screening studies. The two recombinant plasmids were transformed into E.coli BL21(DE3) strains. Then, we demonstrated that concentration of the two strains chloramphenicol growth were 34?g/mL, and we didn't detected an obvious protein band of reporter gene in induced L1-CAT-pET21a/BL21, but detected an obvious protein band of reporter gene in induced L11-CAT-pET21a/BL21 strains.(2) Isolation of enhancer-like sequence-containing strains and identification of enhancer-like sequence vectorsSamples of sewage or soil were collected, and the sample added to medium for mixed bacteria enrichment. Genomic DNA of mixed bacteria was extracted. The best condition of digested genome with Sau3A I was determinated, and digested genomic DNA fragments were inserted into two enhancer-probing vectors to construct gene libraries. Three enhancer-like sequence-containing strains were screened by chloramphenicol. The chloramphenicol concentration were increased by 5 times in growth of ER1-L1-CAT-pET21a/BL21 strain and ER2-L1-CAT-pET21a/BL21 strain by induction of the reporter gene expression; and the chloramphenicol concentration was increased 11 times in growth of ER3-L11-CAT-pET21a/BL21 by induction of the reporter gene expression. The results of restriction enzyme digestion and sequencing showed that ERl-L1-CAT-pET21a and ER2-L1-CAT-pET21a contained insertive sequences, and the sequence of recombinant vectors skeleton had partially gene deleted. Additionally, ER3-L11-CAT-pET21a also contained an insertive fragement and the sequence of recombinant vectors skeleton hadn't been deleted.(3) Analysis of reporter gene expression in enhancer-like sequence-containing strains and Enhancer-liker sequenceThe enhancer-like sequence-containing strains were induced by IPTG and identified by SDS-PAGE. The results showed that L1 1-CAT gene expression was increased by 2.26 times in induced ER3-L11-CAT-pET21a/BL21strain, but report genes expression weren't tested in induced ER1-L1-CAT-pET21a/BL21 strain and ER2-L1-CAT-pET21a/BL21 strain because their vectors had deletion mutants. There were an obvious protein band beteewn 45 kDa and 66.2 kDa in the two induced strains, so the results were the same as the deletion mutant of remobinant vectors. ER1 and ER2 were inserted into L11-pET21a, and the gene expression results showed that L11 protein was increased by 1.78 times and 1.37 times respectively.Analysis of sequences by NCBI showed that:1) ER1 had little nucleotide homology. ER1229-411bp had about 90% homology with a variety of prokaryotes, and ER1412-517bp had the highest homology with hydrophila Aeromonas, accounting for 77%; 2) ER2 had about 100% homology with hydrophila Aeromonas; 3) ER3 had the highest homology with Bacillus cereus, accounting for 71%. Predicted transcription binding sites were showed that these sequences contianed multiple binding sites, and many binding sites were combined with a variety of transcription factors. Analysis of conserved regions showed that ER1 had two conserved regions, and ER3 had one conserved region.(4) Analysis of the functional region of Enhancer-like sequencesBy end-deletion assay, the functional region of ER1 and ER3 were determined. The data demostrate that the enhancing function region of sequence ER1 was about 200bp between 117bp and 317bp, and the enhancing function region of sequence ER3 was about 265bp between lbp and 265bp. The transcription binding sites were predicted by onLane software. The results of ER1117-317 and ER31-265 showed that the two sequences contianed multiple binding sites. The transcription binding sites or their transcription factors of ER31-265 were much more than ER1117-317.Three enhancer-like sequences were screened from the genomic DNA samples. Then, we made an sequences analysis, and the functional region of ER1 and ER3 were determined. Transcription binding sites and their transcription factors of their functional regions were predicted. This study will provide a foundation for the enhancer-like element of theoretial reseach and practical application.