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A Synthetic Biology Approach To Engineered Competency in E. coli. Samantha Simpson Genomics 4/8/09. 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.

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A Synthetic Biology Approach To Engineered Competency in E. coli

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A synthetic biology approach to engineered competency in e coli

A Synthetic Biology

Approach To Engineered

Competency in E. coli

Samantha Simpson

Genomics

4/8/09


Why would you want to insert new dna into e coli

Why Would You Want To Insert New DNA into E. coli?

Insulin Gene Extracted

Plasmid


My focus

My Focus

Insulin Gene Extracted

Plasmid


A closer look at taking up new dna

A Closer Look at Taking Up New DNA


What is competency

What Is Competency?


How does natural competency work

How does natural competency work?


How does comk activate late competency genes

How does ComK activate late competency genes?

ComK

Late Competency Gene

K-box promoter


How are cells made competent

How are cells made competent?

  • Natural Competency in B. subtilis

    • Controlled by ComK

  • Induced Competency in E. coli

Both

48 genes

Microarray

57 genes

K-boxes

1014


Is natural competency feasible in e coli

Is natural competency feasible in E. coli?


Is natural competency feasible in e coli1

Is natural competency feasible in E. coli?

K-Box to gene distance not consistent.


Building a testable system

Building a Testable System

}

600 bps

ComK

ComK

MWM

High Copy

AmpR

1018 bp

506 bp

396 bp

344 bp

298 bp


Building a testable system1

Building a Testable System

RBS

ComK

GFP

pKBox

Low Copy

High Copy

AmpR

KanR


Testing fluorescence

Testing Fluorescence

p < 0.02


Building a testable system2

Building a Testable System

RBS

RBS

ComK

GFP

pKBox

Low Copy

High Copy

AmpR

KanR


Rbs s not consistent

RBS’s: Not Consistent


Promoters consistent

Promoters: Consistent


Building a competent system

Building a Competent System

ComK

Low Copy

KanR


Testing competency

Testing Competency

ComK / Kan

Pellet Cells

RFP / Amp

LB + Kan/Amp


Conclusions

Conclusions

In E. coli:

In B. subtilis:

pKBox + ComK


Acknowledgments

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


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