Construction And Application Of Conditional Expression And Knockout Systems In Escherichia Coli

After the complete of genome sequencing program, researchers focus on the functional genomic study. Using perturbations of DNA, genetic analyses have provided many insights into the functions of proteins encoded by specific genes. However, it can be difficult to study essential genes using these approaches, and many biological processes occur on a fast timescale that precludes study using simple genetic methods. In this thesis, we constructed two different conditional systems for function study.Temperature-sensitive (TS) mutants provide an extremely powerful tool for studying protein function in vivo and in cell culture, which have several unique advantages over such systems, including fast temporal response, high reversibility, and the applicability to any tissue type or developmental stage of an organism. TS mutants are commonly generated by random mutagenesis with a chemical mutagen or ultra violet irradiation, followed by laborious screening of large number of progenies. However, such approaches were suffered from the fact that not all proteins can be generated to TS and the TS mutants are often too leaky to be useful. Based on the fact that buried hydrophobic amino acids are the key to proteins thermo-stability, TS mutants could be generated solely by substitution of the buried hydrophobic residue(s) with Asp. Yeast TS mutants were also generated by fusing protein(s) with a heat-inducible degron. Although these efforts simplify TS mutant creation, the modified peptide sequence of these mutants may affect the normal protein functions or introduce additional function at the temperatures below the non-permissive temperature. Furthermore researchers found interrupting with TS intein, proteins could be converted into TS mutants. However, additional experiments should be done to screen the optimal site(s) for TS intein integration.To avoid above shortcomings, we combined the advantages of temperature-sensitive intein and T7 system, constructed a novel TS T7-expression system to create TS mutants in Escherichia coli. A TS T7-RNA polymerase is generated by interrupting with a TS intein from Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) at position Ala491 and Cys 492, resulting that the gene flanked by T7-promoter and T7-terminator will be transcribed only at the permissive temperature (18℃), not at the restrictive temperature (37℃). The feasibility of this strategy to create TS mutants was detected using lacZ gene and tet gene as target. In vitro T7 RNA polymerase assay indicated the intein insertion at position Ala491 and Cys 492 was effective. In vivo protein expression experiments showed that the target protein could be expressed effectively by TS T7 RNA polymerse at permissive temperature. Cell phenotypes were also correlated with the T7 RNA polymerase activity. Reverse transcriptase polymerase chain reaction (PCR) indicated that the transcription of target gene was controlled by the activity of T7 RNA polymerase. At 18℃, transcripts of T7-promoter controlled gene were at least 85 times more than those at 37℃. In addition, Western blot analysis and enzymatic assay showed large amounts of active His6-tagged protein produced at 18℃but little at 37℃. Furthermore, the shutoff of gene expression was fast and efficient on the shift to the restrictive temperature. And experiments showed that T7-terminator was indispensable for this system, as it could terminate the T7-transcription efficiently in such chromosome harbored T7-expression cassette. This strategy can avoid an extensive, often unsuccessful search mutation in interest gene and the risk of null mutation or expression. It can also avoid the time-consuming work of inserting position selection and amino acid effect detection, but also can convert multiple genes into TS mutants in one cell just by inserting them under a T7-promoter in cluster, which may help us to resolve the novel functions carried out by several proteins. In this T7-system, the expression level at permissive temperature is efficient and quite similar to the system with normal T7-RNA polymerase (with no intein). Therefore besides the creation of TS mutants, this system can be used for high-level expression of heterologous proteins at low temperature, especially proteins that might misfold at higher temperatures. Furthermore, considering T7-terminator can stop the T7-transcription efficiently in vivo, a byproduct of our research is that we can construct the engineered strain in which both T7-RNA polymerase gene and T7-expression cassette are embedded in E. coli chromosome so that antibiotics, the potential contaminants of food or drugs, could be omitted from the medium.Essential genes are of particular interest because they include the core functions upon which basic cellular processes are based, they tend to be widely conserved and serve as a starting point for minimal genome designs. But difficulties in mutating essential genes have impeded their genetic study. Approaches were developed to study essential gene function, such as generation of temperature-sensitive (TS) mutants, controlled expression of target gene with controllable promoter, gene translation interference by antisense RNA or TS suppressor tRNA. However, all these strategies frustrated as no true null mutants could be made, which may result in the possibility of leaky gene expression under non-permissive conditions, the difficulty to design, laborious work, or expression overridden, was also be concerned. Here we constructed another conditional knockout-rescue system based on arabinose-induced Flp/FRT system, which can be used to screen for unknown essential genes from other organims. Two FRT sites and positive/negative markers were integrated into chromosome DNA surrounding the essential gene. And the reporter plasmid containing arabinose-induced FLP gene and GAPDH promoter controlled perspective gene was transformed into this cell. When arabinose added, Flp would recognize and excise the sequences between the FRT sites, leaving one FRT site on chromosome. Cell could grow only when the prospective gene on the plasmid complemented to the deleted gene, as true null mutant generated. System efficiency detection showed that when FLP gene located on plasmid and the FRT sites on chromosomal DNA, induced in M63 medium containing 0.2 % arabinose, the excision efficiency could reach nearly about 100 %. lacZ and dnaE complementation assays after induced with arabinose, the chrosomal gene was deleted and the cell survival depended on the plasmid copy. This is the base of unknown gene screen. As true null mutant are obtained using this method, it will eliminate the leaky possibility of gene expression under non-permissive conditions. This is especially important when genes are analyzed that require only low expression levels for some functions. In addition, before Flp-mediated deletion, the target gene is expressed from its own promoter, which should allow proper regulation. In contrast, the use of a repressible promoter may cause improper expression of the target gene even under permissive conditions and artificial results. In addition, this system does not require much previous knowledge about a particular target gene and it can be used to study those genes that cannot be mutated as TS. Furthermore, multiple prospective genes came from different organisms or DNA libraries can be screened using this system simultaneously. And the complex function of subunits can also be studied by expressing them together. A particularly attractive possibility will be the use of this system to find unknown essential genes of pathogen. As many essential genes are attractive targets for developing new antibacterial agents, a detailed knowledge of them will help researchers to design the inhibitors.Using this system, we detected two different ORF (CP0654 and CP0782) of Chlamydia pneumonia AR39 (C. pneumoniae AR39), which encoding Ribonuclease H (RNase H) homologues. Database search indicated that there are two RNase H encoding gene in C. pneumoniae AR39. Sequence alignments showed that these two homologues both belonged to type 2 RNase H, but CP0654 encoded RNase HII (Cpn-RNase HII) and CP0782 encoded RNase HIII (Cpn-RNase HIII). Complementation assay indicated that Cpn-RNase HII could rescue the two E. coli rnh- knockout mutants, while Cpn-RNase HIII could only complement the rnhA- knockout E. coli. In vitro enzyme activity experiments show neither Cpn-RNase HII nor Cpn-RNase HIII were thermo-stable and their optimum pH values were 9.0 and 10.0, respectively. They needed Mg2+ or Mn2+ in reaction buffer. As to the substrate specificity, Cpn-RNase HII could cleave the 12 bp RNA/DNA substrate at multiple sites, but Cpn-RNase HIII only one. When the DNA-RNA-DNA/DNA chimeric substrate used, cleavage was only observed with Cpn-RNase HII. The different properties of the two proteins imply that Cpn-RNase HII maybe the major RNase H of C. pneumoniae.In a word, these two conditional systems provide us new ideas to regulate the target gene expression, which could be used in different fields.