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Improvement in Electroporation Transformation Efficiency of Gene for Bacillus Subtilis with Trehalos

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Tutor: LuZhaoXin
School: Nanjing Agricultural College
Course: Of Food Science
Keywords: Bacillus subtilis,trehalose,competent,electroporation transformation
CLC: Q78
Type: Master's thesis
Year:  2010
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Abstract:
Genetic transformation has provided an important means of genetic exchange, which has been used for genetic analysis. Bacillus subtilis, a spore-forming gram-positive bacterium, has been developed to lead to transformation with exogenous DNA. Several hundred wild B. subtilis strains have been collected, with the potential to produce more than two dozen antibiotics with an amazing variety of structures and to use for the production of various hydrolyzing enzymes. It is a representative organism of gram-positive bacteria and therefore has different protein expression capability which gram-negative bacteria such as E. coli cannot provide. Genetic transformation provides a modern gene technology for the improvement of industrially important microorganisms by introducing exogenous genes. Such technology can only be realized if the microorganisms are transformable. However, the low competence or unnaturally competence of several Bacillus strains led to development of other strategies involving phage transduction, protoplast fusion the highly versatile electro-transformation method, and the electroporation of protoplast method. One of low efficiency reasons has been reported which was due to a much reduced level of competence, Competence in Bacillus subtilis develops after To, which is the point of transition from exponential growth to stationary phase. This temporal control is initiated by two extracellular peptides:ComX and CSF (competence and sporulation factor). These peptides are released in the medium by growing cells and increase in concentration concomitantly with cell density. However, these methods are difficult to transform natural isolates of Bacillus strains sometimes due to their low genetic competence levels. So, it is important to elaborate on finding the post-exponential phase of Bacillus strains. Other of low efficiency reasons is less the electrical pulse and unsuitable exogenous DNA used to transform, or Bacillus strains relatively thick and dense cell walls and the percentage of cell death by electrical damage. So it is necessary to search for suitable time and power of electroporation and protective agents from electric damage.Trehalose (a-D-glucopyranosyl a-D-glucopyranoside) is a unique sugar capable of protecting biomolecules against environmental stress. It is a stable, colorless, odor-free and non-reducing disaccharide, and is widespread in nature. At first, trehalose was thought to serve as a means of carbohydrate storage but lately it was shown to be produced in response to stress. Studies on the hydration potential of trehalose, compared to other oligosaccharides, demonstrate that trehalose has the highest ability for hydration. This suggests that trehalose may stabilize lipid bilayers by ordering the water molecules around the membrane or by direct interaction with polar bio-molecules as water is removed. Presumably, this is protective action of trehalose may contribute to the damaged cells during particle bombardment. Ideally, improvement of cell survivability will lead to an increase in efficiency.In this study, we examined the effect of trehalose on the electro-transformation efficiencies and cell survivability of Bacillus subtilis 168; so that we can provide a valuable tool for molecular genetic analysis of recalcitrant natural Bacillus strains, which are hard to transform by conventional methods. We have developed a trehalose electroporation method for the improvement of transformation efficiency of Bacillus subtilis by a combination of an appropriate competence phase and high trehalose concentrations in the electroporation medium as well as in the recovery media. This improvement in transformation efficiency is largely attributed to the correct competence phase and the improved cell survivability through protection by trehalose media during electro-transformation.
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