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Cloning of the genes that code for three major subunits of Escherichia coli polymerase III

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

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  1. 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

  2. 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

  3. Department Information • The department is divided into 6 programs • Mikrobiologi • Gerhart’s research focus on: ’riboregulator’ regulatory RNAs in bacteria

  4. Project

  5. Introduction • There exists a very stringent control of DNA replication in bacteria

  6. 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)

  7. DNA polymerase III holoenzyme has a central role in chromosomal replication

  8. DNA polymerase III core is composed of α, ε and θ subunits • and code by dnaE, dnaQ and holE

  9. So we can • mutate negtively control genes • express DNA polymerase III core Test the DNA content

  10. 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

  11. Experiment protocol and result

  12. 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

  13. Primer designing • For each insert: Include translation initiation site ATG • For the first insert: Include the Shine-Dalgarno sequence GGAA

  14. What is Shine-Dalgarno (SD) sequence ?

  15. 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

  16. First several bases of mRNA form a loop

  17. PCR result dnaE PCR product 3500bp

  18. dnaQ PCR product 800bp 700bp

  19. Cleavage of pBAD open-circular supercoiled PvuII EcoRI both linear

  20. Ligation (Ready-to-go T4 DNA lagase)  transformation (TOP-10 chemically competent E.coli)  colony PCR

  21. Acknowledgement • Many thanks to Prof. Gerhart Also thank the member of the lab, especially Klas, Cia, Shiying and Erik

  22. Thank you!

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