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

Project

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

Experiment protocol and result

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

What is Shine-Dalgarno (SD) sequence ?

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

First several bases of mRNA form a loop

PCR result 3500bp dnaE PCR product

800bp 700bp dnaQ PCR product

Cleavage of pBAD PvuII EcoRI both open-circular supercoiled 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

Thank you!