Hi, does anyone khonw which plasmid can we use in cloning in pseudomonas? I have to overexpress two genes in pseudomonas and I don't know which plasmid can replicate and express in pseudomonas.
Thanks
For cloning and overexpressing genes in Pseudomonas, you can use several plasmids that are designed to replicate and express in this genus. Some popular options include the pUCP series, pBBR1MCS series, and pME6000 series, which are shuttle vectors capable of replicating in both E. coli and Pseudomonas. If you need inducible expression, consider plasmids like pJN105 (arabinose-inducible) or pMMB67EH (IPTG-inducible). When choosing a plasmid, it's important to consider factors such as antibiotic resistance markers, copy number, promoter strength, and compatibility with your specific Pseudomonas species. For overexpressing two genes, you might use a single plasmid with two multiple cloning sites or two compatible plasmids. It's recommended to review the literature for the most suitable plasmid for your specific Pseudomonas strain and experimental needs.
To overexpress two genes in Pseudomonas, you can consider using one of the following plasmid vectors:
1. **pUCP18/19 plasmids**: These are broad-host-range plasmids that can replicate and express genes in various Pseudomonas species. They have a high copy number and contain the lac promoter for inducible gene expression.
2. **pJN105 plasmid**: This is another broad-host-range plasmid that can be used for gene expression in Pseudomonas. It has a low copy number and utilizes the arabinose-inducible PBAD promoter.
3. **pME6032 plasmid**: This plasmid is derived from the RSF1010 replicon and can be used for gene expression in Pseudomonas. It has a medium copy number and contains the tac promoter for inducible gene expression.
4. **pVLT33 plasmid**: This plasmid is based on the RK2 replicon and can be used for gene expression in Pseudomonas. It has a medium copy number and contains the Ptrc promoter for inducible gene expression.
When choosing a plasmid, consider the following factors:
- Copy number: Higher copy number plasmids (e.g., pUCP18/19) may lead to higher expression levels, while lower copy number plasmids (e.g., pJN105) may be more stable.
- Promoter: Select a promoter that suits your needs (e.g., lac, PBAD, tac, Ptrc) and allows for inducible expression of your genes.
- Antibiotic resistance marker: Ensure that the plasmid contains an appropriate antibiotic resistance marker for selection in Pseudomonas.
It's recommended to test different plasmid options and monitor the expression levels of your target genes to determine the most suitable plasmid for your specific needs.
you can try it plasmids for cloning in Pseudomonas that are below:
pUCP19 / pUCP24, pBBR1MCS, pMF54, pPSV38 and, of course, some others you could try as well,
but anyway, some main factors should be considered: 1) Compatibility with the host strain, 2) Selectable markers, 3) Copy number, etc.
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