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 Mutagenesis Overexpression Purification 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 500bp--- 900bp--- 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 1 2 3 4 5 6 7 8 9 10 11 12 13

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 Size exclusion chromatograph

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 Collins Group Members: Brett Goldsmith Steve Hunt Alex Kane Bucky Khalap Tatyana Sheps Danny Wan Phil Haralson Yu-Jin Chen John Coroneus 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 IM-SURE/UROP program National Science Foundation Said Shokair Professor Philip Collins Professor Gregory Weiss

17. Questions?