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Broad host-range vector for efficient expression of foreign genes in gram-negative bacteria.
S H Rangwala1, R L Fuchs, D J Drahos
1Monsanto Corporate Research, St. Louis, MO 63198.
Bio/Technology (Nature Publishing Company)
|May 1, 1991
Summary
A new broad-host-range plasmid enables recombinant protein production in diverse Gram-negative bacteria. The recA promoter and T7 g10-L RBS show broad functionality, enhancing gene expression in various Pseudomonas species.
Area of Science:
- Molecular Biology
- Microbiology
- Biotechnology
Background:
- Developing broad-host-range expression systems is crucial for optimizing recombinant protein production.
- The efficiency of bacterial promoters and ribosome binding sites can vary significantly across different host species.
- Identifying versatile genetic elements is key to expanding the utility of microbial hosts.
Purpose of the Study:
- To construct and characterize a broad-host-range expression plasmid.
- To evaluate the functionality of the Escherichia coli recA promoter and bacteriophage T7 g10-L ribosome binding site (RBS) in diverse Gram-negative bacteria.
- To assess the potential of this system for recombinant protein production in alternative hosts.
Main Methods:
- Construction of a broad-host-range plasmid containing the incQ replicon, E. coli recA promoter, and T7 g10-L RBS.
- Cloning of porcine somatotropin (pst) and E. coli beta-galactosidase (lacZ) genes into the expression vector.
- Transformation and gene expression analysis in a panel of Gram-negative bacteria, including E. coli and various Pseudomonas species.
- Induction of gene expression using nalidixic acid.
Main Results:
- The E. coli recA promoter demonstrated functionality across a wide range of Gram-negative hosts, including Pseudomonas species.
- Gene expression was inducible by nalidixic acid in most tested hosts.
- The g10-L RBS significantly increased gene expression (38-70 fold) in two Pseudomonas hosts compared to previous findings in E. coli.
- Expression levels of lacZ were comparable to or higher than those in E. coli in several hosts.
Conclusions:
- The constructed broad-host-range plasmid with the recA promoter and g10-L RBS is a versatile tool for gene expression studies in diverse Gram-negative bacteria.
- The translational enhancer in the g10-L RBS is functional in various bacterial species beyond E. coli.
- This system provides a straightforward method for evaluating alternative bacterial hosts for enhanced recombinant protein production.