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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 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
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? 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
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