T4 DNA ligase gene을 추출하여 vector에 recombination 시켜 E.coli에 주입하여 IPTG induction을 통해 over expression 시켜주고 MBP tag를 이용해 분리해낸 뒤에 activity test를 한 실험 보고서입니다. 각각의 step에 대한 자세한 기술이 있어 기초 생화학 실험의 이해 및 보고서 작성에 큰 도움이 될 것입니다.
DNA ligase is very commonly used proteins both in the cell and laboratory. They can be useful in biotechnology experiments to create recombinant DNA. We have focused on such importance and decided to harvest T4 DNA ligase. One major way to produce a target protein is using recombinant DNA. Recombinant DNA can specifically increase expression of the target gene thus facilitating harvest of our target proteins. The goal of the experiment is attaining T4 DNA ligase showing high activity by constructing a proper recombinant DNA. MBP-T4 DNA ligase with high activity was obtained through the experiment. Recombinant DNA construction turned out successful and efficient.
DNA ligase participates in many roles in cells. DNA ligase forms phosphodiester bond between DNA strands to join them. DNA ligase takes part in replicating genes. Also, DNA ligase acts to repair single-strand and double-strand breaks in cells, thus maintaining gene stability. DNA ligases are used in many laboratories. Purified DNA ligases, such as T4 DNA ligase, are often used to create recombinant DNA.
Table 3. Comparison of concentration measured according to Coomassie blue staining.
Harvested enzyme showed higher activity than commercial T4 DNA ligase- The purified MBP-T4 ligase was tested its activity as in the condition given in Table 1. The gel electrophoresis was done to observe how much activity the enzyme showed. (Figure 8) Harvested enzymes showed quite high activity and ligated the substrate DNA. However, commercial T4 DNA ligase didn’t show good activity even when put excess unit, so the comparison of activity was impossible.
Lehnman, I. R., 1974, Science 186 (4166), pp.790-798
Tabor. Stanley, 2001, Current Protocols in Molecular Biology, Book 1,
Berg. Jeremy Mark, Tymoczko. John L., Stryer. Lubert, 2010, Biochemistry, 7th ed.