Why Would You Want To Insert New DNA into E. coli?
Insulin Gene
ExtractedPlasmid
My Focus
Insulin Gene
ExtractedPlasmid
A Closer Look at Taking Up New DNA
What Is Competency?
How does natural competency work?
How does ComK activate late competency genes?
K-box promoterComK Late Competency Gene
How are cells made competent?
• Natural Competency in B. subtilis– Controlled by ComK
• Induced Competency in E. coli
K-boxes1014
Both48
genes
Microarray57 genes
Is natural competency feasible in E. coli?
Is natural competency feasible in E. coli?
K-Box to gene distance not consistent.
Building a Testable System
AmpR
ComK
High
Copy
MWM
ComK
1018 bp506 bp396 bp344 bp298 bp
600 bps
Building a Testable System
KanR
ComK
Low
Copy
RBSRBS
AmpR
pKBox
High
Copy
GFP
Testing Fluorescence
p < 0.02
Building a Testable System
KanR
ComK
Low
Copy
RBSRBS
AmpR
pKBox
High
Copy
GFP
RBSRBS
RBS’s: Not Consistent
Promoters: Consistent
Building a Competent System
KanR
ComK
Low
Copy
Testing Competency
ComK / Kan Pellet Cells RFP / Amp LB + Kan/Amp
Conclusions
In E. coli: In B. subtilis:
pKBox + ComK
Acknowledgments
• 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