Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector

Abstract

Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and engineer complex artificial metabolic systems. DNA manipulation on a large genome-wide scale is an inevitable challenge, but a necessary tool for synthetic biology. To improve the methods used for the synthesis of long DNA fragments, here we constructed a novel shuttle vector named pGF (plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid pCC1BAC, which can accommodate large DNA molecules, was chosen as the backbone. The sequence of the yeast artificial chromosome (YAC) regulatory element CEN6-ARS4 was synthesized and inserted into the plasmid to enable it to replicate in yeast. The selection sequence HIS3, obtained by polymerase chain reaction (PCR) from the plasmid pBS313, was inserted for screening. This new synthetic shuttle vector can mediate the transformation-associated recombination (TAR) assembly of large DNA fragments in yeast, and the assembled products can be transformed into Escherichia coli for further amplification. We also conducted in vivo DNA assembly using pGF and yeast homologous recombination and constructed a 31-kb long DNA sequence from the cyanophage PP genome. Our findings show that this novel shuttle vector would be a useful tool for efficient genome-scale DNA reconstruction.

Reconstitution and Expression of mcy Gene Cluster in The Model Cyanobacterium Synechococcus 7942 Reveals a Roll of MC-LR in Cell Division

“…Yeast strain, E. coli strain, algae strains, and culture conditions S. cerevisiae strain VL6-48 (MAT alpha,lys2,met14), E. coli strain EPI300 carrying an inducible trfA gene, and the TAR cloning vector pGF were obtained from the Research Group of Systems Virology, State Key Laboratory of Virology of the Wuhan Institute of Virology, Chinese Academy of Sciences (Hou et al 2016, Shang et al 2017b. M. aeruginosa PCC 7806 was purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB) and cultured in BG11 medium with constant illumination at 60 μmol photons m -2 s -1 at 30°C.…”

“…The TAR cloning vector pGF was used to construct the pGF-NSII-mcy cluster (Fig.S1B), the control (Fig.S1C) and the pGF-mcy cluster (Fig.S1D) plasmids(Hou et al 2016, Kouprina and Larionov 2008, Shang et al 2017b). The pGF-NSII-mcy cluster was synthesized based on TAR in yeast via four steps, as outlined in Fig.…”

“…S1C) and the pGF-mcy cluster (Fig. S1D) plasmids (Hou et al 2016, Kouprina and Larionov 2008, Shang et al 2017b). The pGF-NSII-mcy cluster was synthesized based on TAR in yeast via four steps, as outlined in Fig.…”

“…Further MST and 90° angle light scattering experiments proved that MC-LR irreversibly competes with the GTP binding domain of FtsZ and prevents it from polymerization (Fig.7). The results indicated that by competing with the GTP binding site in FtsZ, MC-LR inhibits the formation of FtsZ protofilaments, and resulted in un-divided long filamentous cells in 7942M, which could possibly be one good reason that blooming cyanobacteria gain a competitive edge over their non-blooming counterparts.In conclusion, we reassembled the pGF-NSII-mcy cluster in vitro by TAR cloning and recombined mcy cluster into the NSII site of the Synechococcus 7942 genome to obtain bioactive MC-LR, and proposed the following working model for the competition between GTP and MC-LR to the binding site of FtsZ (Fig.8): Normally, the binding of GTP to FtsZ monomer initiate the polymerization of FtsZ to its dimeric high affinity conformation, the later will then further cooperatively assembled into protofilament and finally leads to cell division; however, in the presence of MC-LR, which irreversibly competing the GTP binding pocket of FtsZ, both monomer and dimer, hold it back from further polymerization and thus results in abnormal cell division inMaterials and MethodsYeast strain, E. coli strain, algae strains, and culture conditions S. cerevisiae strain lys2, met14), E. coli strain EPI300 carrying an inducible trfA gene, and the TAR cloning vector pGF were obtained from the Research Group of Systems Virology, State Key Laboratory of Virology of the Wuhan Institute of Virology, Chinese Academy of Sciences(Hou et al 2016, Shang et al 2017b. M. aeruginosa PCC 7806 was purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB)…”

Genetic Engineering of Bacteriophages Against Infectious Diseases

“…Alternatively, the full-length genomic DNA can be assembled with overlapping genome fragments in vivo through transformation-associated recombination (TAR), which exploits a high level of homologous recombination in the yeast (Jaschke et al., 2012; Ando et al., 2015). The TAR approach has been used to assemble large DNA up to 300 kb in length (Hou et al., 2016; Shang et al., 2017). The overlapping viral fragments were amplified from the genome by PCR, and each adjacent fragment has a homologous sequence overhang.…”