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

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Presentation transcript:

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? K-box promoter ComK Late Competency Gene

How are cells made competent? Natural Competency in B. subtilis –Controlled by ComK Induced Competency in E. coli K-boxes 1014 Both 48 genes Microarray 57 genes

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 bp 506 bp 396 bp 344 bp 298 bp 600 bps

Building a Testable System KanR ComK Low Copy RBS AmpR pKBox High Copy GFP

Testing Fluorescence p < 0.02

Building a Testable System KanR ComK Low Copy RBS AmpR pKBox High Copy GFP RBS

RBS’s: Not Consistent

Promoters: Consistent

Building a Competent System KanR ComK Low Copy

Testing Competency ComK / KanPellet CellsRFP / AmpLB + 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