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