Transcript Cloning of the gene of three major subunits of Escherichia

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
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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
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The department is divided into 6
programs
Mikrobiologi
Gerhart’s research focus on:
’riboregulator’
regulatory RNAs in bacteria
Project
Introduction
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There exists a very stringent control of
DNA replication in bacteria
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Observation:
mutates all the genes that are know
to negatively control replication
DNA content only goes up 1.5 – 2 folds
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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
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DNA polymerase III core is composed
of α, ε and θ subunits
and code by dnaE, dnaQ and holE
So we can
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mutate negtively control genes
express DNA polymerase III core
Test the DNA content
Strategy
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Use pBAD-TOPO vector to insert in
order dnaE, dnaQ, holE, and with very
little spacing inbtween.
PCR outPCR
theout
genes
the genes
the primers
should
carrycarry
different
the primers
should
restriction
sites
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
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For each insert:
Include translation initiation site ATG
For the first insert:
Include the Shine-Dalgarno sequence
GGAA
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What is Shine-Dalgarno (SD)
sequence ?
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dnaE forward: 5′- [P] – CTGACTGCAGGGAATCTGAAGATGTCTGAA
PstI
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dnaE reverse: 5′- TAGAATTCTACCATGGTTAGTCAAACTCCAGTTCCA
EcoRI
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NcoI
dnaQ forward: 5′- TACCATGGAAGTCTGACATAAATGACCGCT
NcoI
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dnaQ reverse: 5′- TAGAATTCTAGGTACCTTATGCTCGCCAGAGGCAAC
EcoRI
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KpnI
holE forward: 5′- TAGGTACCGAGGAGATTAAGAATG
KpnI
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holE reverse: 5′- TAGAATTCTTATTTAAGTTTGGGCT
EcoRI
First several bases of mRNA
form a loop
PCR result
dnaE PCR product
3500bp
dnaQ PCR product
800bp
700bp
Cleavage of pBAD
open-circular
supercoiled
PvuII
EcoRI
both
linear
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Ligation
(Ready-to-go T4 DNA lagase)
 transformation
(TOP-10 chemically competent E.coli)
 colony PCR
Acknowledgement
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Many thanks to Prof. Gerhart
Also thank the member of the lab,
especially Klas, Cia, Shiying and Erik
Thank you!