why would you want to insert new dna into e. coli? insulin gene extracted plasmid
TRANSCRIPT
![Page 1: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/1.jpg)
![Page 2: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/2.jpg)
Why Would You Want To Insert New DNA into E. coli?
Insulin Gene
ExtractedPlasmid
![Page 3: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/3.jpg)
My Focus
Insulin Gene
ExtractedPlasmid
![Page 4: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/4.jpg)
A Closer Look at Taking Up New DNA
![Page 5: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/5.jpg)
What Is Competency?
![Page 6: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/6.jpg)
How does natural competency work?
![Page 7: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/7.jpg)
How does ComK activate late competency genes?
K-box promoterComK Late Competency Gene
![Page 8: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/8.jpg)
How are cells made competent?
• Natural Competency in B. subtilis– Controlled by ComK
• Induced Competency in E. coli
K-boxes1014
Both48
genes
Microarray57 genes
![Page 9: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/9.jpg)
Is natural competency feasible in E. coli?
![Page 10: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/10.jpg)
Is natural competency feasible in E. coli?
K-Box to gene distance not consistent.
![Page 11: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/11.jpg)
Building a Testable System
AmpR
ComK
High
Copy
MWM
ComK
1018 bp506 bp396 bp344 bp298 bp
600 bps
![Page 12: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/12.jpg)
Building a Testable System
KanR
ComK
Low
Copy
RBSRBS
AmpR
pKBox
High
Copy
GFP
![Page 13: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/13.jpg)
Testing Fluorescence
p < 0.02
![Page 14: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/14.jpg)
Building a Testable System
KanR
ComK
Low
Copy
RBSRBS
AmpR
pKBox
High
Copy
GFP
RBSRBS
![Page 15: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/15.jpg)
RBS’s: Not Consistent
![Page 16: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/16.jpg)
Promoters: Consistent
![Page 17: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/17.jpg)
Building a Competent System
KanR
ComK
Low
Copy
![Page 18: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/18.jpg)
Testing Competency
ComK / Kan Pellet Cells RFP / Amp LB + Kan/Amp
![Page 19: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/19.jpg)
Conclusions
In E. coli: In B. subtilis:
pKBox + ComK
![Page 20: Why Would You Want To Insert New DNA into E. coli? Insulin Gene Extracted Plasmid](https://reader035.vdocuments.us/reader035/viewer/2022062217/56649ca65503460f94968f5c/html5/thumbnails/20.jpg)
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