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Creation of an endA mutant strain in Pseudomonas aeruginosa PAO1 using gene replacement

dc.contributor.authorOlsen, Cassie J., author
dc.contributor.authorKarkhoff-Schweizer, Roxann R., author
dc.description.abstractEndonuclease I is an enzyme encoded by the endA gene. This nuclease degrades double stranded DNA. Many Escherichia coli common laboratory strains contain a mutation in the endA gene that inactivates the DNA-specific endonuclease I. A mutation in this gene greatly increases plasmid DNA yields in such E. coli strains as well as improves the quality of DNA that is isolated. The purpose of this research is to create an endA mutant strain in Pseudomonas aeruginosa PAO1 using gene replacement, thereby leading to the development of a useful laboratory Pseudomonas strain for use as a cloning strain. To accomplish this, chromosomal DNA from P. aeruginosa PAO1 was isolated, and the endA gene was then amplified by PCR using specific primers designed to the flanking upstream and downstream sequence of the endA coding region. The resulting amplified 1100 bp DNA fragment containing the endA gene was cloned into pCR2.1. This newly created plasmid was named pCR2.1-endA. In order to create an insertionally inactivated endA gene, a GmR encoding cassette from pPS856 needed to be inserted into the SalI sites of the cloned endA gene. The pCR2.1-endA plasmid was digested using SalI restriction enzyme. A 4500 bp SalI fragment of pCR2.1-endA was isolated and then religated by T4 DNA ligase. The new plasmid created was called pCR2.1-endASalID. This plasmid was digested with SalI, and blunt ends were created with T4 DNA polymerase. Inactivation of the endA gene was accomplished by insertion of a blunt-ended, GmR encoding gene into the blunt-ended SalI site of the endA coding sequence. The resulting recombinant plasmid was called pCR2.1-endASalID(Gm1). A 1700 bp HindIII x PstI DNA fragment from pCR2.1-endASalID(Gm1), containing the insertionally inactivated endA gene, was isolated and cloned into the similarly digested pEX18Ap plasmid.
dc.description.awardHigh Honors.
dc.format.mediumStudent works
dc.publisherColorado State University. Libraries
dc.relation.ispartof2004 Projects
dc.rightsCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see
dc.subject.lcshPseudomonas aeruginosa
dc.subject.lcshRestriction enzymes, DNA
dc.titleCreation of an endA mutant strain in Pseudomonas aeruginosa PAO1 using gene replacement
dcterms.rights.dplaThis Item is protected by copyright and/or related rights ( You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s)., Immunology, and Pathology Medicine and Biomedical Sciences


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