Download Inserting a Competency Regulatory Gene into E. coli

Why Would You Want To Insert
New DNA into E. coli?
Insulin
Gene
Extracted
Plasmid
My Focus
Insulin
Gene
Extracted
Plasmid
A Closer Look at Taking Up
New DNA
What Is Competency?
How does natural competency
work?
How does ComK activate late
competency genes?
ComK K-box promoter
Late Competency Gene
How are cells made competent?
• Natural Competency in B. subtilis
– Controlled by ComK
K-boxes
1014
Both
Microarray
48
57 genes
genes
• Induced Competency in E. coli
Is natural competency feasible
in E. coli?
Distance to Kbox in E. coli (bps)
22000
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
Distance to Kbox in B.subtilis (bps)
22000
24000
26000
Is natural competency feasible
in E. coli?
Distance to Kbox in E. coli (bps)
22000
20000
K-Box to gene distance
not consistent.
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
Distance to Kbox in B.subtilis (bps)
22000
24000
26000
Building a Testable System
600 bps
ComK
ComK
MWM
1018 bp
506 bp
396 bp
344 bp
298 bp
Building a Testable System
RBS
ComK
pKBox
GFP
Testing Fluorescence
12
10
rlu/OD
8
6
p < 0.02
4
2
0
GFP
pKBox:B0032:GFP
pKBox:B0032:GFP +ComK
Building a Testable System
RBS
ComK
RBS
pKBox
GFP
RBS’s: Not Consistent
210
180
150
rlu/OD
120
90
60
30
0
Promoters: Consistent
210
180
150
rlu/OD
120
90
60
30
0
Building a Competent System
ComK
Testing Competency
ComK / Kan
Pellet Cells
RFP / Amp
LB + Kan/Amp
Conclusions
In E. coli:
pKBox +
ComK
In B. subtilis:
Acknowledgments
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Davidson Research Initiative
National Science Foundation
Howard Hughes Medical Institute
James G. Martin Genomics Program
Dr. Heyer, Dr. Campbell and Dr. Denham
Fellow lab workers
Friends and Family