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

Chengxi ShiMolecular Biotechnology and

BioinformaticsUppsala 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

PvuIIEcoRI

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!