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Optimization of a protoplast transformation method for Bacillus Subtilis, Bacillus megaterium, and Bacillus Cereus by a plasmid pHIS1525.SplipA
Högskolan i Borås, Institutionen Ingenjörshögskolan.
Högskolan i Borås, Institutionen Ingenjörshögskolan.
2009 (Engelska)Konferensbidrag, Poster (med eller utan abstract) (Övrigt vetenskapligt)
Abstract [en]

During the past years of gene cloning studies, Escherichia coli has always been a foremost host cell for exogenous genes expressions owing to its high level of protein production and excretion. However, problems relating to low level of extracellular production of some proteins specially the accumulation of cloned proteases within the cells have moved the attentions from E.coli to bacilli bacteria such as B. megaterium, B.subtilis, and B.cereus due to their secretion ability of many different enzymes. Bacillus megaterium is widely used for high-level expression of heterologous proteins with little or no degradation. Bacillus subtilis is a naturally competent host cell for uptake of exogenous DNA, resulting in attractive industrial applications. Bacillus cereus has sporulation capability which makes it suitable for several industrial uses. A conventional approach for transferring DNA into protoplasts or intact cells of bacillus bacteria is chemical transformation, using chemicals through chilling and then shock-heating of the suspension of cells to induce reversible permeabilization of the cell membrane to make it possible for the external DNA to enter into the cells. In most cloning experiments, the transformation with plasmid DNA is performed using Polyethylene glycol (PEG)-induced competence cells. In this study, a PEG-induced protoplast transformation protocol was developed for three different bacillus strains of Bacillus megaterium ATCC®14945, Bacillus Subtilis ATCC®6051, and Bacillus Cereus ATCC®14579. In all cases a plasmid pHIS1525.SPlipA, well working vector in B.megaterium, was applied. Protoplasts were formed in RHAF medium after treating the cells with lysozyme. Two factors, the incubation time and the lysozyme concentration have been found to play the most important role in effective protoplast formation. These two factors were further optimized in this study to elaborate a chemical transformation procedure which can possibly work for other bacillus strains as well. The optical density (A420) and the number of colony-forming units (CFUs) were determined to find the optimal conditions for each strain. The results indicate that PEG-induced protoplast transformation is a sufficient technique when using a plasmid pHIS1525.SPlipA in Bacillus genus.

Ort, förlag, år, upplaga, sidor
2009.
Nyckelord [en]
bacillus megaterium, bacillus subtilis, bacillus cereus, peg-induced protoplast transformation
Nyckelord [sv]
Energi och material
Nationell ämneskategori
Annan industriell bioteknik
Identifikatorer
URN: urn:nbn:se:hb:diva-6324Lokalt ID: 2320/5987OAI: oai:DiVA.org:hb-6324DiVA, id: diva2:887011
Konferens
BioMicroWorld2009, III International Conference on Environmental, Industrial and Applied Microbiology, 2-4 december, Lissabon, Portugal
Tillgänglig från: 2015-12-22 Skapad: 2015-12-22 Senast uppdaterad: 2017-10-05Bibliografiskt granskad

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Feuk-Lagerstedt, ElisabethSárvári Horváth, Ilona

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