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Evidence for stable transformation of Pseudomonas aeruginosa with a pUC-based plasmid
Yongsoon Kim1, Dale A Webster, Benjamin C Stark
1Biology Division, Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA.
Abstract:
Pseudomonas aeruginosa was transformed with pUC8:16, a pUC-based plasmid bearing the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). Transformation was initially indicated by an increase in ampicillin resistance from 1500 to 2500 mg l(-1). Presence of the plasmid in P. aeruginosa was confirmed by amplification of a portion of vgb from and detection of VHb in the transformant but not the untransformed host. Southern blot analysis further indicated that pUC8:16 existed as an autonomous plasmid rather than integrated into the chromosome of the P. aeruginosa transformant.
Insights
Pseudomonas aeruginosa was successfully transformed with a plasmid carrying the Vitreoscilla (bacterial) hemoglobin gene (vgb). The transformants showed increased ampicillin resistance and confirmed VHb presence, indicating successful gene transfer.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Pseudomonas aeruginosa is an opportunistic pathogen.
- Bacterial hemoglobins (bHb) offer potential for enhanced oxygen transport.
- Genetic manipulation of P. aeruginosa is crucial for biotechnological applications.
Purpose of the Study:
- To introduce and express the Vitreoscilla hemoglobin gene (vgb) in Pseudomonas aeruginosa.
- To confirm the presence and functional expression of the bacterial hemoglobin in the host.
- To analyze the state of the introduced plasmid within the P. aeruginosa transformant.
Main Methods:
- Transformation of P. aeruginosa with the pUC8:16 plasmid carrying the vgb gene.
- Assessment of ampicillin resistance to indicate successful transformation.
- PCR amplification to confirm the presence of the vgb gene.
- Detection of Vitreoscilla hemoglobin (VHb) in transformants.
- Southern blot analysis to determine plasmid status (autonomous vs. integrated).
Main Results:
- Transformation resulted in a significant increase in ampicillin resistance.
- The vgb gene was successfully amplified from transformant DNA.
- Vitreoscilla hemoglobin (VHb) was detected in the transformed P. aeruginosa.
- Southern blot analysis confirmed the plasmid remained autonomous, not integrated into the chromosome.
Conclusions:
- The pUC8:16 plasmid containing the vgb gene can be successfully introduced and maintained in Pseudomonas aeruginosa.
- The transformed P. aeruginosa expresses functional Vitreoscilla hemoglobin.
- The plasmid exists as an autonomous entity within the host, facilitating further genetic studies and applications.