![]() Western blotting further proved the single band at the same position. SDS-PAGE showed that relative molecular mass (Mr ) of the purified product was consistent with the expected value. ![]() Restriction enzyme digestion and sequencing demonstrated that the recombinant plasmid was valid. The expression of the CR2-Fc fusion protein was detected and confirmed by SDS-PAGE and Western blotting. The ones with stable expression of the fusion protein were obtained by means of G418 selection. After verified by restriction enzyme digestion and sequencing, the recombinant plasmid was transfected into CHO K1 cells. The extracellular domain of human CR2 and IgG1 Fc were respectively amplified, ligated and inserted into the eukaryotic expression vector PCI-neo. To construct a eukaryotic expression vector containing human complement receptor 2 (CR2)-Fc and express the CR2-Fc fusion protein in Chinese hamster ovary (CHO) cells. Li, Xinxin Wu, Zhihao Zhang, Chuanfu Jia, Leili Song, Hongbin Xu, Yuanyong The rapid construction of a BAC-based expression vector facilitates Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by λ-RED-mediated recombination to obtain the BAC-based expression vector. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.Ĭhen, Chao Zhao, Xinqing Jin, Yingyu Zhao, Zongbao Kent Suh, Joo-Wonīacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. Finally, both vectors will be useful for the large-scale gene expression industry. It also provides a method for recombination at a specific chromosomal location. Our work provides an effective transformation system for homologous and heterologous gene expression and gene knockout in T. Additionally, we measured the exo-1,4-β-glucanase activity of the recombinant cells. The applicability of both vectors was tested by first generating the expression vectors pWEF31-red and pWEF32-red and then detecting the expression of the DsRed2 gene in T. On the other hand, pWEF31 undergoes random recombination. As a result, pWEF32 easily undergoes homologous recombination. The difference between pWEF31 and pWEF32 is that pWEF32 has two longer homologous arms. reesei with Agrobacterium-mediated transformation. The two newly constructed vectors can be efficiently transformed into T. Both vectors possess the hygromycin phosphotransferase B gene expression cassette and the strong promoter and terminator of the cellobiohydrolase 1 gene (cbh1) from T. We report the construction of two filamentous fungi Trichoderma reesei expression vectors, pWEF31 and pWEF32. Plate out the suspension on a LB agar plate containing the appropriate antibiotic.Resuspend cell pellet with the rest SOC medium in the tube by pipetting.Spin down briefly and remove most supernatants.Incubate for 1 hour at 37☌ and shake vigorously.Place the tubes immediately on ice for at least 2 min.Heat shock the cells for 45 sec to 2 min at 42☌.Transformation of plasmid DNA to competent E.
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