Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid

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