| Cloning and mutagenesis in vitro of _amylase gene were studied and four main contents included. The new methods to identify _amylase activity and its producing bacteria, and the preparation of high efficiency competence cell were reported. Three _amylase genes were cloned from Bacillus subtilis,Xanthomonas campestrisand Aspergillus oryzae.Two mutant genes with high activity was reported by using the methods of mutagenesis in vitro .1.A new method to identify _amylase activity and its producing bacteria: The blue complex was formed by unspecific adsorption, after mixing starch and trypan_blue. The adsorption weakened when the starch was hydrolyzed to small molecular by _amylase, and the trypan_blue was released inside the hydrolyed zone. The starch around the zone which was not hydrolyzed adsorbed free trypan-blue so that the colour of medium became bluer than that of place in hydrolyzed zone. A transparent halo was formed in this place. This screening medium was designated as LBSP. Its " optimum concentration of trypan blue was from 0.005% to 0.01%(w/v), the optimum volume was around 15ml/per petri dish (080mm). LBSP medium was easy to use and can be sterilized by 121掳C for 30 minutes. The sensitivity and application of trypan blue method was the same as the idione starch method. The advantage was that the trypan-blue was harmless to a-amylase and its producing bacteria. 2.The preparation of high efficiency competence cell: The preparation of high efficiency competence cell was one of the most important techniques in molecular biology. It was necessary for gene library construction and transformation with enzyme ligased products. There were many factors to affect the preparation of competant cells. The affected factors of preparation of competence cells and transformation process had been studied. The results were summarized. The different media showed different transformation efficiencies. High concentration of salts such as CaCl2and MnCl2 reduced significantly the efficiency of competence cells. The most important factor was the start pH value, incubation time and the cleaness of the container used. The optimum conditions for the preparation of competence cells with high efficiency were the following: (1) start pH value in medium was 7.0. (2) the cultivation time was 1.2-1.5h and OD800 from0.053to0.l10. (3) the container used should be washed three times by deionized water.The time and temperature of heat shock, delayed after heat shock, the cleaness oftransformation container, solution for dissoving plasmid, the size and concentration of plasmid were also studied. The results showed that the concentrations of plasmid were in direct proportion to the transformation efficiency. The transformation efficiency dropped when the plasmid concentration was over the optimum level, but the numbers of transformants were the same. The suitable concentration of plasmid should be used in transformation, When the size of plasmids was getting larger the transformation efficiency was getting lower. The competent cells with high efficiency could be used with large plasmids and the normal competent cells used with small plasmids. The clearer the transformation container was, the higher the transformation efficiency was. Different transformation methods showed different results and efficiencies for common, simple and fast methods were from 5xl08tol05transformants /per ug plasmid DNA. 3.The cloning of a-amylase gene: The methods of shotgun, PCR and RT-PCR were selected to clone a-amylase genes from Bacillus subtilis HN503,Xanthomonas campestris pv. campestris 8004 and Aspergillus oryzae,HN504 The recombinant plasmid with cloned gene was designated as pHN504, pHN503 and pHN8004. The shotgun method was only used with B. subtilis HN503.The optimun conditions for total DNA Extraction, enzyme digest, ligation and transformation were studied The results showed that the most important factor was transformation efficiency. Since the concentration of ligation products was low in common. There would be no transformants and...
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