Mark M. Doolittle

www.linkedin.com/in/mark-doolittle-68784b82

Chelmsford, MA (c: 978-***-****/h: 978-***-****) Skills & Talents

Environmental Molecular-Microbiologist / Bench Scientist experienced in conducting studies focused on the detection, identification, quantification, removal, and source ID and tracking of microbes (e.g. bacteria, phage, viruses, fungi) in water, food, and tissue matrices using qPCR, ddPCR, fluorescent-labelled tracers / probes. Possess expertise in culture of bacterial (free-living & biofilm), fungal, and mammalian cells including infection by bacteriophage & viruses. Proficiency in molecular cloning with recombinant expression vectors, protein characterization, and assay development, validation, and implementation. Developed expertise in SOP development, lab and field validation studies including data validation, sampling, and quality assurance project plans.

Education

University of Massachusetts / Boston (ABD - Ph.D.) Environmental Sciences GPA 3.6 (1989-1994)

• US-Canada Fulbright Pre-doctoral Research Scholar in Dr. D. Caldwell’s Biofilm Studies Laboratory at University of Saskatchewan in Saskatoon, CA. Project proposal funded to study: Visualization and quantification of infection of bacterial biofilms by lytic bacteriophage.

• Dissertation Project: Effect of Inorganic and Organotins on the Interaction of Bacteriophage with Free-Living & Biofilm Bacteria

Vanderbilt University Molecular Biology Dept. – Predoctoral Studies GPA 3.6 (1985-1987)

• Research: Interaction of T4 phage DNA adenine methylase (dam) and gene 69 on DNA replication, gene expression, and phage genome methylation. Effects of methionine deprivation on mammalian HeLa cell metabolism and genomic DNA methylation patterns. University of Tennessee / Knoxville – M.S. Microbiology GPA 3.6 (1981-1985)

• Thesis: Characterization of DNA Sequence Specificity / Preference of Wildtype (Dam) and Mutant Hypermethylating (Damh) Methylases of Phage T2 & T4 & Wildtype Host Ecodam Methylase. University of Massachusetts / Dartmouth, B.S. in Biology GPA 3.2 (1973-1977)

• Scored 96% (Ranked No.1 UG) on National Organic Chemistry Exam

• Ranked No. 1 as UG in UG/Grad Developmental Biology Lecture/Lab Course Professional Experience

Environmental Consultant (self employed), 01/21-present Bench Scientist, Task Lead of Applied Research Group, Liberty Biosecurity LLC

(EdenRoc Sciences Expeditionary Laboratory), Worcester, MA, 01/18-10/20

Developed SOPs and conducted laboratory performance tests of novel proprietary bio-products for microbial biofilm removal. Biofilms cultured in CDC Dynamic Flow / Drip Flow Reactors and 96-well plates. Mechanism of Action (MOA) and Stability Studies conducted upon bio-products. Biofilm removal rates quantified by Crystal Violet Stain Binding/Elution +/- Blotting or SEM visualization after Fixation, CPD, & Sputter Coating. Bench Scientist, Dermalytica LLC, Beverly, MA, Startup Company, 4/16 - 12/17

Technical lab support for development of biological (e.g. enzymatic) treatments for dermatological maladies caused by Propionibacterium acnes. Analyzed select gene sequence variants for cloning. Performed functional phenotypic (e.g. enzymatic activity; viability assays) and molecular analyses of proteins extracted and purified from transformed bacterial clones containing recombinant DNA constructs.

Enzyme characterization and Minimum Inhibition Concentration (MIC) Determinations Bacteriologist, Mass. Dept. Public Health, HS LI, Food Outbreak Section, 5/14 - 1/15

Completed MassDPH validation and implementation of FDA Method of Combined MPN Culture- Amplification and qPCR assay for detection and quantification of Vibrio parahaemolyticus contamination in raw shellfish (i.e. oysters)

Strain maintenance ID confirmation of reference stocks and isolates.

Acquired CDC certification by demonstrating proficiency for Salmonella and E.coli strain ID by Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) utilizing PCR and CE.

Inter-platform & inter-method performance evaluations and validations for qPCR assays. Seasonal Engineer Aide, Mass. Conservation & Recreation Department, 5/13 - 9/13

Recreational Water Sample collections at DCR beaches and transport to analytical labs.

Mapped GPS coordinates of all Mass DCR Beach Water Sampling Sites using Google GIS Mapper and developed 2014 Beach Water Quality Report describing factors affecting Water Quality including potential human and animal sources. Microbiologist, USEPA New England Regional Lab, ESAT Contract, 12/02 - 4/13

As ESAT Contract Sr. Molecular Biologist for TechLaw Inc. & Lockheed Martin Information Technologies supervised 3 junior biologists while conducting 12 national and regional water quality and fecal source tracking studies for EPA R1’s Office of Environmental Measurement & Evaluation (OEME) using validated Enterococcus and FRNA coliphage rtPCR/RT-PCR and culturable fecal source tracking assays.

Developed high-throughput water sample processing protocols on the Roche MagNA Pure LC automated magnetic bead nucleic acid isolation platform to rapidly process samples for PCR analysis using the Roche LightCycler, ABI 7500, and Cepheid SmartCycler PCR platforms.

Lead EPA Region I OEME’s development, validation, and implementation of EPA Method 1609/1611 for Quantification of Enterococcus DNA by TaqMan PCR as part of Multi-Lab Validation Study and Single Lab Validations conducting PCR Sample Hold Time Study. Environmental Analyst, Massachusetts Conservation & Recreation Dept., 4/01 - 11/02

Coordinated DCR Beaches Water Quality Testing / Sampling Program providing technical oversight of contract analytical lab. Conducted sanitary survey to identify potential fecal sources impacting storm water runoff and beach water quality. Developed annual reports summarizing trends in water quality and identifying potential sources of fecal contamination at DCR beaches.

Performed environmental review of “in-house” engineering projects and permit applications for projects that impacted DCR properties requiring meetings and communications with staff members and consultants for power utilities (i.e. Mirant) and USEPA, DEP, DPH, CZM, & ACOE. Environmental Analyst, Mass. DEP Wall Experiment Station (NEIWPCC), 9/97 - 3/01

Analyzed 300 MassDEP New England Groundwater Study (EPA) water samples submitted by five states for Rotavirus, Hep A, Norwalk, and Enteroviruses (e.g. poliovirus, coxsackie virus) by RT-PCR & Tissue Culture Infectious Dose (TCID50) Assays in addition to bacterial and coliphage fecal indicators (culture / plaque assays).

Microbiologist, Gillette Corp. Personal Care Products Division, 9/93 – 4/96

Preservative challenge testing of products, raw material and finished product bioburden testing, contaminant ID testing using selective media and Biolog ID platform. Performed product performance comparison testing for Product Claims Department Research Technician III, MIT Brain & Cognitive Science Department, 2/89 - 2/90

Viral & Bacterial Expression Vector Cloning of Nerve Growth Factor (NGF), Restriction Mapping, Mammalian Cell Culture (primary & “immortal” cell lines) and Transfection, Large Scale DNA & RNA Isolation and Southern / Northern Hybridization Analysis of E-Gel (Native & Glyoxal) Blots Areas of Vocational Interest

Molecular/Enzymatic/Chemical / Biochemical Assay Development, Validation, Food & Water Quality, Genetics, Molecular Biology, Microbiology, Pathogens, Virology, Indicators, Human Health, Forensics, Environmental Monitoring, ddPCR, CRISPR, Phage Therapies & Tools Technical Skills

Growth of bacterial biofilms in CDC Dynamic Biofilm Bioreactors and Drip Flow Reactors under batch and continuous flow conditions for testing efficacy of products upon biofilms.

Isolation, concentration, and purification of bacteriophage strains from cultured lysates.

Fluorescent labelling of phage particles and utilization in detection and eradication of planktonic and biofilm bacteria.

Mammalian Tissue Culture/Roller Bottle Culture for Virus TCID Quantitation

Molecular biological, chemical/biochemical, analytical, and troubleshooting skills for method / protocol / process / product / policy development, optimization, validation, and implementation.

Design of PCR primers & Hydrolysis, Hybridization, Molecular Beacon probes

Cloning in M13 and T7 phage and T7-promoter plasmid expression vectors

Miniprep/Maxiprep DNA isolation/purification, electroporation, transfection

Optimization of media / conditions favoring recombinant gene expression and production

Selective & Non-Selective Aerobic and Anaerobic culture of BSL-1&2 bacteria and fungi

qPCR, RT-PCR, MLVA, Sanger Dideoxy Chain Termination & Next-Gen Sequencing

Microscopy (SCLM, PC, Fluor, TEM, SEM), Cloning

Cepheid SmartCycler, GeneXpert, ABI-7500/7700/7900, Roche LightCycler, MagNA Pure LC

Nucleic Acid & Protein Electrophoresis & Staining: Capillary, Agarose, Polyacrylamide

Soluble and Inclusion Body Concentrated Protein Isolation & Purification

Native and SDS PAGE Electrophoretic Protein Analysis & Characterization

ELISA, Western, Northern, & Southern Blotting / Hybridization

Chromatography: Affinity, Size Exclusion, Anion / Cation Exchange, HPLC, GC-FID

Fatty Acid Methyl Ester Analysis (FAME GC-FID analysis)

Heterotrophic Bacteria ID by Biolog™ and BD Vitek ID Systems

Software: Word, Excel, PowerPoint; LIMS, Statistica, Primer Express, BLAST (NCIB)

Strong leadership, supervisory and communication skills

Advanced technical writing and publication / presentation skills.

Antibiotic Resistance Testing, Microbial Stock Maintenance

Mechanical and electrical aptitude with automated instrumentation (e.g. plate readers) Professional / Technical Training / Workshops

ASM Workshop: Statistics for Clinical Laboratory Rapid Testing. ASM Workshop: PFGE & DNA Fingerprinting. LRN Workshop: Identification of Biological Warfare Agents. ATCC Workshop: Preservation Technology & Fungal Infections. SCCWRP Rapid Methods Study, Orange County Sanitation District, CA 2005

Meetings & Presentations

UNC Water Institute Water Microbiology Conference, May 2017 American Society of Microbiologists (ASM) 1994, 2006, 2009, 2014, 2016 EPA National Beaches Meetings, Huntington Beach, CA 2006, 2009 National Annual Quantitative PCR Meeting, San Diego, CA. 2009, 2013, 2015 Publications

Chandler, J.C., A. Pérez-Méndez, J. Paar, M. Doolittle, B. Bisha and L.D. Goodridge (2017) Field testing and evaluation of the inhibitory effects of environmental waters on the detection of F-RNA coliphages using an anionic-exchange resin method. J. Virological Methods 239:9-16 Paar, J., M.M. Doolittle, M. Varma, S. Siefring, K. Oshima and R.A, Haugland (2015) Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences. J. Microbiol. Methods 112: 28-35

Pérez-Méndez A. T., J. C. Chandler, J. Paar, M. Doolittle, E. Bouthiette, B. Bisha1, S. M. Coleman, and L. D. Goodridge (2013) Validation of a rapid resin-based method for concentration and further detection of F-RNA coliphages in different water sources of the New England Region., ASM Annual Meeting Poster Presentation

Chern, E., Siefring, S., Paar, J., Doolittle, M. and R. Haugland. (2011) Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes. L. Appl. Microbiol. 52, 298– 306)

Wymer, L., Oshima, K., Paar III, J., Doolittle, M., Lavender, J., Varma, M. and R. Haugland. (2010) Effects of holding time, storage, and the preservation of samples on sample integrity for the detection of fecal indicator bacteria by quantitative Polymerase Chain Reaction (qPCR)-based Assays., EPA/600/ R-10/150 December 2010

M. Yasuda, J. Paar, M. Doolittle, J. Brochi, O.C. Pancorbo, R.J. Tang, R.E. Stoner, M.P. Shiaris. Enterococcus species composition determined by capillary electrophoresis of the groESL gene spacer region DNA. Water Research 44:3982 (2010)

Doolittle, M.M., J.J. Cooney and D.E. Caldwell. (1994) Lytic infection of Escherichia coli biofilms by bacteriophage T4. Can. J. Microb. 41:12

Doolittle, M.M., J.J. Cooney and D.E. Caldwell. (1993) Tracing the interaction of bacteriophage with bacterial biofilms using fluorogenic and chromogenic probes. J. Ind. Microb. 16:331 Doolittle, M.M. and K. Sirotkin (1988) Bacteriophage T2 and T4, dam+ and damh and Ecodam+ methylation: preference at different sites. Biochimica et Biophysica Acta 949:240 Professional References

Dr. Oscar Pancorbo, Ph.D., Station Chief, Mass DEP Wall (Lawrence) Experiment Station, 37 Shattuck Road, Lawrence, MA. email: adqu4p@r.postjobfree.com; tel. 978-***-**** x51314 Dr. Jean Tang, Ph.D., Scientist, Molecular Studies Division Manager, Mass DEP Wall

(Lawrence) Experiment Station, 37 Shattuck Road, Lawrence, MA. email: adqu4p@r.postjobfree.com; tel. 978-***-****

Dr. Jim Wang, Ph.D., CEO, Dermalytica, LLC, STEM Center, Endicott College, Beverly, MA. email: adqu4p@r.postjobfree.com tel. 508-***-****

Mr. Jack Paar, Task Order Project Officer, USEPA OEME Ecosystems Assessment Division, New England Regional Laboratory, 11 Technology Drive, North Chelmsford, MA 01863 email: adqu4p@r.postjobfree.com; tel. 617-***-**** (receptionist tel. 617-***-****) Mr. Paul DiPietro, (Retired) Former Head of Water Resources Section, Engineering Division, former Metropolitan District Commission (MDC), currently Massachusetts Department of Conservation & Recreation (Mass DCR), 7 Maple Road, Medford, MA, tel. 617-***-****

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