CLONING, INDUCTION and HETEROLOGOUS EXPRESSION

The production of thermozymes of industrial interest is usually achieved through over-expression of the corresponding genes in mesophilic bacterial models or in eukaryotic hosts used as cell factories. However, only a fraction of active-form thermozymes with industrial applications can be overproduced in these hosts.

Thermus spp. is one of the most extreme groups of thermophilic bacteria, and their genes can encode enzymes of great biotechnological potential. T. thermophilus is an alternative cell factory for the production of thermozymes and a useful host for the genetic manipulation of plasmids. The high growth rates and the high culture yields make this thermophile one of the most promising models of subrogate cell factories for the overproduction of proteins (1, 2).

Biotools offers a portfolio of tools, including vectors and close kits, for the expression and thermo-stabilisation of proteins in different systems. Our technology provides an innovative and highly efficient method of transferring DNA fragments to vectors adapted to grow at high temperatures (1-6). Our vectors offer some of the following features:

• Shuttle Vectors with bifunctional promoters, active in E. coli and T. thermophilus.
  
  
• The presence of a kanamycine cassette (kat) for the thermostable selection of entry clones
  
  
• The presence of the Pnar promoter for nitrate-induced expression
  
  
• The presence of the nitrate reductase operon (nar operon) which controls the respiratory metabolism in hosts during their adaptation to anaerobic growth
  
  
• High transformation efficiency

References:

1. De Grado M. et al. (1999). Plasmid 42: 241-5.
2. Hidalgo A. et al. (2004). Appl Environ Microbiol 70: 3839-44.
3. Ramírez S. et al. (2000). J Bacteriol 182: 2179-83.
4. Zafra O. et al. (2002). FEBS Lett 523: 99-102.
5. Moreno R. et al. (2003). Plasmid 49: 2-8.
6. Moreno R. et al. (2005). Appl Environ Microbiol 71: 591-3.

Related Products

1. DNA Amplification
2. dNTPs
3. Primers and Probes
4. PCR Enhancer
5. Buffers and Solutions
6. DNA and RNA Purification
7. Electrophoresis Reagents