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Mutagenesis and Overexpression of DNase for Single Molecule Studies

Mutagenesis and Overexpression of DNase for Single Molecule Studies. Denise Der IM-SURE Program 2007 Mentor: Professor Philip Collins Collaborator: Professor Gregory Weiss Graduate Students: John Coroneus, Issa Moody, Jorge Lamboy. Background.

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Mutagenesis and Overexpression of DNase for Single Molecule Studies

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  1. Mutagenesis and Overexpression of DNase for Single Molecule Studies Denise Der IM-SURE Program 2007 Mentor: Professor Philip Collins Collaborator: Professor Gregory Weiss Graduate Students: John Coroneus, Issa Moody, Jorge Lamboy

  2. Background • Single molecule streptavidin attached to carbon nanotube • Attachment was nonspecific • EDC/NHS functionalized carbon nanotube • Tetrameric streptavidin • Four lysines per monomer Figure from http://chem.ps.uci.edu/~gweiss/research.htm EDC = N-ethyl-N’-(3-dimethyl aminopropyl) carbodiimide NHS = N-hydroxysuccinimide

  3. Goal • Site-specific protein attachment via cysteine-maleimide chemistry • Significance: To monitor proteins in real-time, to elucidate kinetic information such as the turnover rate, and to see if the positioning of the attachment affects the performance of nano biosensor

  4. Background • DNase domain of colicin E9 • Previously studied • Monomer • No cysteine residues Figure from the protein data bank online

  5. Methodology Overview Purification Overexpression Mutagenesis Activity Assay Attachment Biosensor Detector

  6. Site-Specific Mutations Two internal mutations: replacing a serine with a cysteine Two external mutations: inserting a cysteine at the N-terminus or C-terminus

  7. Quikchange

  8. Verification 1 2 3 4 5 6 7 8 9 10 11 12 13 Lane 1: 100 bp ladder Lanes 2-3: colony PCR of C-30 mutant Lanes 4-5: miniprep DNA Lanes 6-7: colony PCR of C-30 mutant Lanes 8-13: colony PCR of C-49 mutant 900bp--- 500bp---

  9. Overexpression • Lane 1: molecular weight ladder • Lane 2: post-lysis cell pellet • Lane 3: post-lysis supernatant • Lane 4: pre-lysis supernatant

  10. Flowthrough  Wash  Elution 1  Elution 2

  11. Sample Protein Gel Flowthrough Wash Elution1 of C30S mutant

  12. After Dialysis against Water Lane 1: 1 kb ladder Lane 2: miniprep DNA Lane 3: miniprep DNA + DNase

  13. Arising Problem Sizeexclusionchromatograph

  14. Mass Spectrum Expected kDa: 15.11 kDa Actual kDa: 15.104 kDa Impurity at 11.278 kDa

  15. Future Work • To continue working on purifying the DNase mutants • Adding protease inhibitor and inducing for less time • To attach to a functionalized nanotube • To measure the conductance of single molecule

  16. Acknowledgements IM-SURE/UROP program National Science Foundation Said Shokair Professor Philip Collins Professor Gregory Weiss • Weiss Group Members: • John Coroneus • Issa Moody • Jorge Lamboy • Michael Todhunter • Calvin Kong • Sarah Kiehna • Lucie Lee • Sudipta Majumdar • Agi Hajduczki • Ryan Lin • Cathie Overstreet • Juan Diaz • Glenn Eldridge • Collins Group Members: • Brett Goldsmith • Steve Hunt • Alex Kane • Bucky Khalap • Tatyana Sheps • Danny Wan • Phil Haralson • Yu-Jin Chen • John Coroneus

  17. Questions?

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