Mutagenesis and Overexpression of DNase for Single Molecule StudiesDenise DerIM-SURE Program 2007Mentor: Professor Philip CollinsCollaborator: Professor Gregory WeissGraduate Students: John Coroneus, Issa Moody, Jorge Lamboy

BackgroundSingle molecule streptavidin attached to carbon nanotubeAttachment was nonspecificEDC/NHS functionalized carbon nanotubeTetrameric streptavidinFour lysines per monomer

Figure from http://chem.ps.uci.edu/~gweiss/research.htmEDC = N-ethyl-N-(3-dimethyl aminopropyl) carbodiimide NHS = N-hydroxysuccinimide

GoalSite-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

BackgroundDNase domain of colicin E9Previously studiedMonomerNo cysteine residues

Figure from the protein data bank online

Methodology OverviewMutagenesisOverexpressionPurificationActivity AssayAttachmentBiosensorDetector

Site-Specific MutationsTwo internal mutations: replacing a serine with a cysteineTwo external mutations: inserting a cysteine at the N-terminus or C-terminus

Quikchange

Verification500bp---900bp---Lane 1: 100 bp ladderLanes 2-3: colony PCR of C-30 mutantLanes 4-5: miniprep DNALanes 6-7: colony PCR of C-30 mutantLanes 8-13: colony PCR of C-49 mutant1 2 3 4 5 6 7 8 9 10 11 12 13

OverexpressionLane 1: molecular weight ladderLane 2: post-lysis cell pelletLane 3: post-lysis supernatantLane 4: pre-lysis supernatant

Flowthrough Wash Elution 1 Elution 2

Sample Protein GelFlowthrough Wash Elution1 of C30S mutant

After Dialysis against WaterLane 1: 1 kb ladderLane 2: miniprep DNALane 3: miniprep DNA + DNase

Arising ProblemSize exclusion chromatograph

Mass SpectrumExpected kDa: 15.11 kDaActual kDa: 15.104 kDa

Impurity at 11.278 kDa

Future WorkTo continue working on purifying the DNase mutantsAdding protease inhibitor and inducing for less timeTo attach to a functionalized nanotubeTo measure the conductance of single molecule

AcknowledgementsCollins Group Members: Brett GoldsmithSteve HuntAlex KaneBucky KhalapTatyana ShepsDanny Wan Phil HaralsonYu-Jin ChenJohn CoroneusWeiss Group Members: John CoroneusIssa MoodyJorge LamboyMichael TodhunterCalvin KongSarah KiehnaLucie LeeSudipta MajumdarAgi Hajduczki Ryan Lin Cathie OverstreetJuan Diaz Glenn Eldridge IM-SURE/UROP programNational Science FoundationSaid ShokairProfessor Philip CollinsProfessor Gregory Weiss

Questions?

Colors too dark?Add in a another little cartoon key here on the sidePut in protein gelDoubly charged ion