Polyhydroxybutyrate-enhanced transformation of log-phase Escherichia coli.
Transformation of Escherichia coli plays an important role in recombinant DNA technology. Most current transformation protocols require that the cells be treated to attain a particular physiological state known as "competence," and this makes transformation procedures lengthy and arduous. Here we describe a protocol for transforming log-phase E. coli using dimethyl sulfoxide (DMSO) solutions of poly-(R)-3-hydroxybutyrate (PHB) to facilitate the transfer of plasmid DNA into cells, and certain reagents and temperature shocks to promote DNA uptake. The protocol was optimized using factorial design techniques across variables that included PHB molecular weight and concentration, DMSO concentration, monovalent and divalent salts, glucose, cold and heat shocks, cell density, and pH. Using 10 ng DNA, the optimized protocol produces approximately 1000 colony-forming units (CFUs) from 100 microL early log-phase cell culture or approximately 300 CFU from a 21-24 h single colony, sufficient for many applications. The total volume of the transformation reaction mixture is only 150 microL suggesting that the procedure may be adapted for use in microplates or automated transformation technologies.
Michigan State University, East Lansing, MI 48824, USA.
Charles J
CJ
Addison
S Haihua
SH
Chu
Rosetta N
RN
Reusch
D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006885:Hydroxybutyrates; D007202:Indicators and Reagents; D010957:Plasmids; D011091:Polyesters; D011995:Recombination, Genetic; D014169:Transformation, Bacterial; D000094122:Polyhydroxybutyrates
D004269:DNA, Bacterial; D006885:Hydroxybutyrates; D007202:Indicators and Reagents; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates