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Zymornonas rnobilis for Ethanol Production
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
Hideshi Yanase, Nobuo Kato, Kenzo Tonomura
We constructed a shuttle vector between Z. mobilis and E. coli by fusion of a native plasmid of Z. mobilis with an E. coli vector plasmid. First, we found that Z. mobilis ATCC 10988 contains three small plasmid species (1.7, 2.5, and 3.9 kb) [28]. Misawa et al. [29] reported that the 1.7-kb plasmid can be subdivided into three species. Since these plasmids were all cryptic, the resistant genes for Tcr and Cmr coded on the E. coli vector plasmid pACYC184 (4.0 kb) were selected as markers of the shuttle vector. Hybrid plasmids were constructed by insertion of the 3.9-kb plasmid pZM3 or the 2.5-kb plasmid pZM2 of Z. mobilis ATCC 10988 into pACYC184. The plasmid pZM3 was ligated with pACYC184 at an EcoRl site, Bgl II and BamHl sites, or Hindlll site to form hybrid plasmids, pZA31, pZA32, and pZA33, respectively [30]. These hybrid plasmids, 7.9 kb in length, have one gene that can be selected for as an antibiotic marker, and a few useful restriction enzyme recognition sites. To construct a more useful vector with two antibiotic marker genes, pZA323 was constructed by the insertion of the Tcr gene from E. coli vector plasmid pBR322 (Apr, Tcr; 4.6 kb) into pZA32, and also pZA22 was constructed by the joining of pZM2 to pACYC184 at a Ava I site [31]. Figure 1 shows restriction maps of these hybrid plasmids.
Production of VNPs, VLPs, and Chimeras
Published in Nicole F Steinmetz, Marianne Manchester, Viral Nanoparticles, 2019
Nicole F Steinmetz, Marianne Manchester
Baculoviruses are insect viruses with large double-stranded DNA genomes that can accommodate multiple foreign genes of interest. Baculoviruses can be grown in cultured insect cells, such as Spodoptera frugiperda (Sf) lines 9 and 21 and Trichoplusia ni cells. The gene(s) of interest are typically placed under control of the viral polyhedrin or plO promoters; both give rise to high yields of gene product. A limitation of the system is the large size of the baculovirus genome; unique restriction sites are not available for simple cloning. Alternative strategies employ the insertion of the gene of interest in a shuttle vector and its introduction into the expression vector by recombination. There are many commercial reagents available to facilitate baculovirus expression; however, compared with E. coli- or yeast-based expression systems these methods are relatively time-consuming and cumbersome. It typically takes a minimum of several days to a few weeks until the recombinant virus is constructed and a molecular clone is isolated.
Sustainability and Development of Industry 5.0
Published in Pau Loke Show, Kit Wayne Chew, Tau Chuan Ling, The Prospect of Industry 5.0 in Biomanufacturing, 2021
Hui Shi Saw, Abdul Azim bin Azmi, Kit Wayne Chew, Pau Loke Show
Interestingly, the choice of expression system is no longer limited to a single host system. This is made possible by the use of a shuttle vector, which is a cloning vector that can replicate in two widely different species. Even though gene cloning techniques are often transferable, they might be limited for some organisms, and therefore requires the advantage of shuttle vector to carry out initial cloning, commonly using E. coli before attempting on other host organisms (Dale 2010). This follows the concept of gene insertion into an E. coli plasmid with an origin of replication that enables the function in another host, as presented in Bacillus subtilis for the protein secretory expression (Guo et al. 2014).
Production of recombinant lethal factor of Bacillus anthracis in Bacillus subtilis
Published in Preparative Biochemistry & Biotechnology, 2021
Mahboobeh Gholami, Majid Moghbeli, Farshid Kafilzadeh, Mohammad Kargar, Mariam Bikhof Torbati, Ashkan Tavizi, Sally Bellevile, Javad Hatami, Zahra Eslami
The Escherichia coli and Bacillus species are widely used to produce recombinant proteins at laboratory and industrial scale. Since cultivation of these bacteria is simple and cost-effective, they are good candidates to be used for mass production.[21,22] In addition to being proper expression hosts, shuttle vectors have high functionality in producing the recombinant proteins. A shuttle vector is a vector which can replicate in at least two different host species. Thus, the genes inserted in the shuttle vector can be evaluated in two different cell types. The main advantage of these vectors is that they can be manipulated in a common host such as E. coli and then can be transformed to cells with slower growth or more difficult genetic manipulation, such as B. subtilis.[23]Bacillus subtilis, a rod-shaped, Gram-positive and facultative aerobe bacterium, is the most genetically available organism and has been identified as a safe production host. On the other hand, B. subtilis has an inherently high secretory capacity for secretion of heterologous proteins into the culture medium in high amounts.[24] Advantages of protein secretion into the media are: correct folding, inhibition of the formation of inclusion bodies, and the ease of protein separation from the culture medium.[25,26] Although the lef gene has been expressed in E. coli, expression and purification of lef gene from B. subtilis has not been very successful. This study aimed to clone and produce lef gene of B. anthracis toxin in B. subtilis WB600 using shuttle vector PHT43. B. subtilis WB600 works as the expression host due to owing a secretion system which does not have the challenge of inclusion bodies formation. Eventually, the lef gene identification was confirmed by dot-blotting and enzyme linked immunosorbant assay (ELISA) and Electrochemiluminescence (ECL) tests.