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Cloning of the genes that code for three major subunits of Escherichia coli polymerase III. Chengxi Shi Molecular Biotechnology and Bioinformatics Uppsala University winter,2005. About Research Training. Period: November to December
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Cloning of the genes that code for three major subunits of Escherichia coli polymerase III Chengxi Shi Molecular Biotechnology and Bioinformatics Uppsala University winter,2005
About Research Training • Period: November to December • Institute: Department of Cell & Molecular Biology • Work time: 9 am to 5 pm at weekdays • Supervisor: Prof. Gerhart Wargner
Department Information • The department is divided into 6 programs • Mikrobiologi • Gerhart’s research focus on: ’riboregulator’ regulatory RNAs in bacteria
Introduction • There exists a very stringent control of DNA replication in bacteria
Observation: mutates all the genes that are know to negatively control replication DNA content only goes up 1.5 – 2 folds • A thought: the number of DNA polymerase molecules in the cell is limiting (usually 8-10 molecules per cell)
DNA polymerase III holoenzyme has a central role in chromosomal replication
DNA polymerase III core is composed of α, ε and θ subunits • and code by dnaE, dnaQ and holE
So we can • mutate negtively control genes • express DNA polymerase III core Test the DNA content
Strategy • Use pBAD-TOPO vector to insert in order dnaE, dnaQ, holE, and with very little spacing inbtween. PCR out the genes the primers should carry different restriction sites PCR out the genes the primers should carry different restriction sites
Overview design primers re-streak bacteria stain PCR plasmid miniprep PCR product purification enzyme cleavage enzyme cleavage gel extraction gel extraction ligation transform into competent cells colony PCR test
Primer designing • For each insert: Include translation initiation site ATG • For the first insert: Include the Shine-Dalgarno sequence GGAA
dnaE forward: 5′- [P] – CTGACTGCAGGGAATCTGAAGATGTCTGAA PstI • dnaE reverse: 5′- TAGAATTCTACCATGGTTAGTCAAACTCCAGTTCCA EcoRI NcoI • dnaQ forward: 5′- TACCATGGAAGTCTGACATAAATGACCGCT NcoI • dnaQ reverse: 5′- TAGAATTCTAGGTACCTTATGCTCGCCAGAGGCAAC EcoRIKpnI • holE forward: 5′- TAGGTACCGAGGAGATTAAGAATG KpnI • holE reverse: 5′- TAGAATTCTTATTTAAGTTTGGGCT EcoRI
PCR result dnaE PCR product 3500bp
dnaQ PCR product 800bp 700bp
Cleavage of pBAD open-circular supercoiled PvuII EcoRI both linear
Ligation (Ready-to-go T4 DNA lagase) transformation (TOP-10 chemically competent E.coli) colony PCR
Acknowledgement • Many thanks to Prof. Gerhart Also thank the member of the lab, especially Klas, Cia, Shiying and Erik