ABSTRACT
Life system has created rich and colorful genes, to protect the inheritance and prosperity after more than 4 billion years of natural evolution. However, the natural evolution is an extremely slow process, and the origin and evolution of new gene with new function often takes millions of years. Therefore, natural evolution alone cannot meet the rapid development of industrial biotechnological production needs. Using synthetic biology techniques, researchers can design and synthesize new genes based on the known enzyme catalysis mechanism and protein structure according to industrial production requirements, and create various biochemical reactions that cannot be catalyzed by natural living organisms. Although the new gene design technology shows exciting application prospects, there are now still many scientific and technological challenges, such as low success rate of design, low catalytic activity and high synthesis cost. With the rapid development of synthetic biology, the design, transformation, synthesis, screening and other technologies will be integrated into a mature technological process for the new gene design.
ABSTRACT
Synthetic biology is an emerging discipline, which aims at creating artificial lives or remolding the present organisms to generate new features. To achieve these goals, synthetic biologists need to design and synthesize new genes, pathways, modules or even whole genomes. As these enabling technologies (e.g. gene synthesis, DNA assembly and genome editing) are very important for the progress of synthetic biology, we will focus on the development of these technologies in this review.
ABSTRACT
This work studied the design, construction, and cleavage analysis of zinc finger nucleases (ZFNs) that could cut the specific sequences within microphthalmia - associate transcription factor (mitfa) of zebra fish. The target site and ZFPs were selected and designed with zinc finger tools, while the ZFPs were synthesized using DNAWorks and two-step PCR. The ZFNs were constructed, expressed, purified, and analyzed in vitro. As expected, the designed ZFNs could create a double-stand break (DSB) at the target site in vitro. The DNAWorks, two-step PCR, and an optimized process of protein expression were firstly induced in the construction of ZFNs successfully, which was an effective and simplified protocol. These results could be useful for further application of ZFNs - mediated gene targeting.
ABSTRACT
AIM:To synthesize the human platelet factor-4(hPF4) gene with a convenient and effective approach, and high express the hPF4 gene in E. coli BL21 (DE3). METHODS: According to the primary structure of hPF4, the nucleotide sequence was synthesized using touch-down PCR method. The resultant gene fragment containing EcoR Ⅰ and Xho Ⅰ overhangs at 5' and 3' ends was cloned into the expression vector pGEX-4T-1 to construct the recombinant plasmid pGEX-4T-1-hPF4,which was then transformed into the E. coli strain BL21 (DE3). RESULTS: hPF4 gene was successfully synthesized by touchdown PCR method. A fusion protein composed of glutathione S-transferase (GST) and the recombinant hPF4 was expressed in BL21(DE3) by IPTG induction. The expression level of the fusion protein in E. coli was about 30% of the total cellular protein. CONCLUSION: Touch-down PCR may provide a convenient and effective approach to obtain other target genes. The expressed fusion protein forms the inclusion bodies, providing sufficient material for further purification and biological activities process.
ABSTRACT
A simple protocol for rapid assembly of chemically synthesized deoxyoligonucleotides into double stranded DNA is described. Several parameters of a ligation-free method were investigated to allow efficient assembly of a large number of oligonucleotides into double stranded DNA by polymerase chain reaction. Synthesis of a 701 bp DNA was carried out in a single reaction by assembling 28 oligonucleotides designed with partial overlaps at complementary ends. An estimate of error rate was made by sequencing several independent clones of the synthesized DNA.