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A Synthetic Biology Approach To Engineered Competency in E. coli PowerPoint PPT Presentation


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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

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


What Is Competency?


How does natural competency work?


How does ComK activate late competency genes?

ComK

Late Competency Gene

K-box promoter


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?

K-Box to gene distance not consistent.


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 System

RBS

ComK

GFP

pKBox

Low Copy

High Copy

AmpR

KanR


Testing Fluorescence

p < 0.02


Building a Testable System

RBS

RBS

ComK

GFP

pKBox

Low Copy

High Copy

AmpR

KanR


RBS’s: Not Consistent


Promoters: Consistent


Building a Competent System

ComK

Low Copy

KanR


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


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