Mutagenesis and Mutagenesis and Overexpression of DNase for Overexpression of DNase for

Single Molecule StudiesSingle Molecule Studies

Denise DerDenise DerIM-SURE Program 2007IM-SURE Program 2007

Mentor: Professor Philip CollinsMentor: Professor Philip CollinsCollaborator: Professor Gregory WeissCollaborator: Professor Gregory Weiss

Graduate Students: John Coroneus, Issa Graduate Students: John Coroneus, Issa Moody, Jorge LamboyMoody, Jorge Lamboy

BackgroundBackground Single molecule Single molecule

streptavidin attached to streptavidin attached to carbon nanotubecarbon nanotube

Attachment was Attachment was nonspecificnonspecific EDC/NHS functionalized EDC/NHS functionalized

carbon nanotubecarbon nanotube Tetrameric streptavidinTetrameric streptavidin Four lysines per Four lysines per

monomermonomer

Figure from http://chem.ps.uci.edu/~gweiss/research.htm

EDC = N-ethyl-N’-(3-dimethyl aminopropyl) carbodiimide NHS = N-hydroxysuccinimide

GoalGoal

Site-specific protein attachment via Site-specific protein attachment via cysteine-maleimide chemistry cysteine-maleimide chemistry

Significance: To monitor proteins in real-Significance: To monitor proteins in real-time, to elucidate kinetic information such time, to elucidate kinetic information such as the turnover rate, and to see if the as the turnover rate, and to see if the positioning of the attachment affects the positioning of the attachment affects the performance of nano biosensorperformance of nano biosensor

BackgroundBackground

DNase domain of DNase domain of colicin E9colicin E9 Previously studiedPreviously studied MonomerMonomer No cysteine residuesNo cysteine residues

Figure from the protein data bank online

Methodology OverviewMethodology Overview

Mutagenesis OverexpressionPurification

Activity AssayAttachmentBiosensorDetector

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

QuikchangeQuikchange

VerificationVerification

500bp---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 mutant

1 2 3 4 5 6 7 8 9 10 11 12 13

OverexpressionOverexpression

Lane 1: molecular Lane 1: molecular weight ladderweight ladder

Lane 2: post-lysis cell Lane 2: post-lysis cell pelletpellet

Lane 3: post-lysis Lane 3: post-lysis supernatantsupernatant

Lane 4: pre-lysis Lane 4: pre-lysis supernatantsupernatant

Flowthrough Wash Elution 1 Elution 2

Sample Protein GelSample Protein Gel

Flowthrough Wash Elution1 of C30S mutant

After Dialysis against Water

Lane 1: 1 kb ladderLane 2: miniprep

DNALane 3: miniprep

DNA + DNase

Arising ProblemArising Problem

Size exclusion chromatograph

Mass SpectrumMass Spectrum

Expected kDa: 15.11 kDaActual kDa: 15.104 kDa

Impurity at 11.278 kDa

Future WorkFuture Work

To continue working on purifying the To continue working on purifying the DNase mutantsDNase mutants Adding protease inhibitor and inducing for Adding protease inhibitor and inducing for

less timeless time

To attach to a functionalized nanotubeTo attach to a functionalized nanotube To measure the conductance of single To measure the conductance of single

moleculemolecule

AcknowledgementsAcknowledgements

Collins Group Members: Brett Goldsmith

Steve HuntAlex Kane

Bucky KhalapTatyana Sheps

Danny Wan Phil HaralsonYu-Jin Chen

John Coroneus

Weiss Group Members: John Coroneus

Issa MoodyJorge Lamboy

Michael TodhunterCalvin Kong

Sarah KiehnaLucie Lee

Sudipta MajumdarAgi Hajduczki

Ryan Lin Cathie Overstreet

Juan Diaz Glenn Eldridge

IM-SURE/UROP programNational Science FoundationSaid ShokairProfessor Philip CollinsProfessor Gregory Weiss

Questions?