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

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

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

11. 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

13. Experiment protocol and result

14. 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

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

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

17. dnaE forward: 5′- [P] – CTGACTGCAGGGAATCTGAAGATGTCTGAA PstI dnaE reverse: 5′- TAGAATTCTACCATGGTTAGTCAAACTCCAGTTCCA EcoRI NcoI dnaQ forward: 5′- TACCATGGAAGTCTGACATAAATGACCGCT NcoI dnaQ reverse: 5′- TAGAATTCTAGGTACCTTATGCTCGCCAGAGGCAAC EcoRI KpnI holE forward: 5′- TAGGTACCGAGGAGATTAAGAATG KpnI holE reverse: 5′- TAGAATTCTTATTTAAGTTTGGGCT EcoRI

18. First several bases of mRNA form a loop

19. PCR result 3500bp dnaE PCR product

20. 800bp 700bp dnaQ PCR product

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

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

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

24. Thank you!