The Methodology Study Of DNA Synthesis And Microorganisms Staining

Synthetic biology, a new idea coming from engineering science, aims at building new biological components and even creatures through synthesizing or changing biological genomes. One of the important tasks of the synthetic biology is to create new low toxic or nontoxic vaccines, while the transformation of low pathogenic microbial genome is essential. During the process of synthetic genome, in vitro DNA fragment synthesis technology is an effective mean to realize the de novo synthesis and directional transformation of synthetic biology. These methods are based on two basic methods including concatenative of DNA ligase and the PCR technology. All these methods are widely used nowadays. To cure humAn diseases, a large number of pathogenic bacteria are nurtured in different kinds of experiments. A large amount of accidents were reported that laboratories mishandled deadly germs over the past decade. Moreover, the leaks of contaminated waste and dropped containers with cultures of bacteria and viruses are invisible and often ignored. So, to improve the bio-safety level, the leakage of pathogenic micro-organism needs for our prompt attention. Thus, an indicator is in need to prevent the bacteria from escaping the specified areas.Our study gives a new method called "High Fidelity DNA Unidirection Synthesis Method". HFUS method mainly contains Phusion DNA polymerase, the BsrDI restriction enzymes and λ exonuclease. By using the same system at different temperatures, HFUS methods will conjunction one positive single-strand DNA and several reverse single-stranded DNA through one by one and eventually synthesized target DNA fragment. The most important advantages of HFUS method is that the size of the synthesized oligo DNA fragments can be more than150nt. In the first step of the core cycle, Phusion DNA polymerase makes up DNA double chain at72℃. Then in the next step, BsrDI restriction endonuclease recognised terminal restriction enzyme recognition site of double stranded R1at50℃. After the specific digestion, the5’ end phosphorylation of the DNA reverse chain are exposed. At the last step of the cycle, Lamda exonuclease can specific recognise and digest of the5’ phosphorylation end of DNA reverse chain at37℃. At last positive single DNA strains are produced. The next cycle starts with seq_f-r1fragment and its downstream sequence can be complementary pairing with seq_r23’ end sequence. After both DNA fragment being engaged to each other, DNA polymerase extend them to double stranded DNA, the product is seq_f-rl-r2.Here, we designed several oligonucleotide fragments, and tested each fragment of oligonucleotide ratio, the amount of enzyme, the enzyme reaction time and reaction parameters. We final synthesis DNA fragments about171bp,340bp,480bp. This study explores a new method of the synthesis DNA fragment, successfully realized the rapid synthesis of480bp long DNA fragment, and provides a new idea for DNA synthesis method.Here, we also investigated the possibility of dying bacteria by using FD&C Yellow No.5(sunset yellow, c16H10N2Na2O7S2).In our first step, a colored LB culture media, with the addition sunset yellow, was used to nurture Escherichia coli strain DH5a while gently coloring the bacteria. The shape, size and traits of the culture can be vividly observed. Further more, it is important to test the dying ability of sunset yellow in culturing pathogenic bacteria. E.coli0157:H7and Shigella flexneri O301were cultivated while marked the culture mixture by sunset yellow. Colony count assays were performed to determine the effects of sunset yellow on bacterial viability. E. coli DH5a cells were serially diluted to into3dilutions(10-5,10-6,10-7). Colony counting was shown to be successful at determining whether the sunset yellow dye inhibits bacterial growth. Alternatively, the number of bacterial colonies turbidity readings of a10-hour E. coli DH5aculture were taken using a spectrophotometer at a wavelength of600nm. This method of staining with sunset yellow was a comprehesive, effective, steady, straightforward and safe way to mark some types of microorganisms. In summary, we describe a novel method by using a colored culture during nurture some dangerous microorganism in laboratories, while dying microorganisms gently and effectively. we are sure the application of bacterial vital staining will be widely used.