Related Experiment Videos
Effects of the par locus on the growth rate and structural stability of recombinant cells
1Microbiology Graduate Group, School of Medicine, University of California at Davis 95616.
Abstract:
A decreased growth rate of recombinant cells is observed when a cloned gene protein encoded in a multicopy plasmid is induced from a strong promoter. This negative effect on the cell growth rate may be the consequence of alternate use or reallocation of energy, precursor metabolites, and protein synthesizing machinery. In order to analyze the causes for this adverse effect, we have studied how genetic elements present in multicopy plasmids affect the growth of recombinant Escherichia coli (K12 delta H1 delta trpEA). Turning on of the PL promoter, whether or not protein-coding sequences were present downstream of the promoter, decreased the growth of the recombinant cells by 15-50%. This result suggests that the essential factor(s) for cell growth may be sequestered by the genetic elements present in the plasmids and thus may cause the decreased growth. The negative effect of the de-repressed PL promoters on cell growth was partially reversed when the par sequence from pSC101 was inserted into the same plasmid. In each case when the plasmid carried only the PL promoter, the PL promoter followed by the N-terminal part of the protein-coding region, or the PL promoter followed by complete sequences encoding a protein, the par sequence exhibited a positive effect on the growth rate of the recombinant cells, which usually grow at a slower rate than the host cell.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Recombinant cell growth slows when strong promoters on multicopy plasmids are induced. Adding a par sequence partially reverses this negative effect, improving growth rates in engineered Escherichia coli.
Area of Science:
- Molecular Biology
- Biotechnology
- Microbial Genetics
Background:
- Recombinant cell growth is often inhibited when expressing foreign genes from strong promoters on multicopy plasmids.
- This growth defect is hypothesized to result from the diversion of cellular resources like energy and protein synthesis machinery.
Purpose of the Study:
- To investigate the impact of genetic elements on multicopy plasmids on the growth rate of recombinant Escherichia coli.
- To identify specific plasmid components responsible for reduced cell growth.
Main Methods:
- Engineered recombinant Escherichia coli (K12 delta H1 delta trpEA) strains carrying multicopy plasmids with varying genetic elements.
- Induction of the PL promoter, with and without downstream protein-coding sequences.
- Assessment of cell growth rates under different plasmid configurations.
- Introduction of the par sequence from pSC101 to evaluate its effect on growth.
Main Results:
- Induction of the PL promoter, regardless of downstream sequences, decreased recombinant cell growth by 15-50%.
- This suggests that plasmid genetic elements sequester essential cellular factors, impairing growth.
- The inclusion of the par sequence partially reversed the negative growth effect.
Conclusions:
- Plasmid-borne genetic elements, particularly strong promoters, significantly impede recombinant Escherichia coli growth.
- The par sequence demonstrates a protective effect, partially mitigating growth inhibition caused by plasmid burden.
- Understanding these interactions is crucial for optimizing recombinant protein production.