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

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

Page 1: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid.
Page 2: 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

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My Focus

Insulin Gene

ExtractedPlasmid

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A Closer Look at Taking Up New DNA

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What Is Competency?

Page 6: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid.

How does natural competency work?

Page 7: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid.

How does ComK activate late competency genes?

K-box promoterComK Late Competency Gene

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How are cells made competent?

• Natural Competency in B. subtilis– Controlled by ComK

• Induced Competency in E. coli

K-boxes1014

Both48

genes

Microarray57 genes

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Is natural competency feasible in E. coli?

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Is natural competency feasible in E. coli?

K-Box to gene distance not consistent.

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Building a Testable System

AmpR

ComK

High

Copy

MWM

ComK

1018 bp506 bp396 bp344 bp298 bp

600 bps

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Building a Testable System

KanR

ComK

Low

Copy

RBSRBS

AmpR

pKBox

High

Copy

GFP

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Testing Fluorescence

p < 0.02

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Building a Testable System

KanR

ComK

Low

Copy

RBSRBS

AmpR

pKBox

High

Copy

GFP

RBSRBS

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RBS’s: Not Consistent

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Promoters: Consistent

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Building a Competent System

KanR

ComK

Low

Copy

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Testing Competency

ComK / Kan Pellet Cells RFP / Amp LB + Kan/Amp

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Conclusions

In E. coli: In B. subtilis:

pKBox + ComK

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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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