Medical Industrial

Sankalp Gupta, Ph.D.

** ******* **, *** # ***, Wakefield, MA 01880

Mobile phone: 201-***-****

E-mail: ***************@*****.***

I have more than 14 years of scientific experience in planning, designing, managing and executing experiments in the fields of functional biochemistry, single molecule microscopy, large scale fermentation, molecular biology & DNA protein interactions. I want to use my scientific experience in the areas of recombinant DNA technology, single molecule analysis, enzymology and in vitro assay development. My experiences range from single molecule DNA replication experiments to 6000 liters large fermentation. I have worked in prestigious research institutes like Memorial Sloan Kettering Cancer Center, New York and Harvard Medical School, Boston with distinct colleagues and teams. I was the first student of my PhD advisor and helped to set up the new lab working on DNA protein interactions in M. tuberculosis before moving to New York to work in DNA replication without any prior working experience and was successful in publishing two back-to-back papers. Then I completely changed my field by joining another lab to do single molecule experiments where we were successful for the first time in the field in demonstrating DNA replication of damaged and undamaged DNA templets in small flow chambers under ultra microscope. Then finally I took up a new challenge in an industrial setting to improve bio- ethanol production in as large fermenters as 6000 liters. We also worked towards establishing gene editing system involving CRISPR technology in new fungal systems. I am not intimidated by new challenges but I work hard to excel in new settings and use my previous experience in solving new problems. My referees will be more than happy to recommend me in any settings to work hard and come up with solutions. I hold US green card therefore I have full work authorization in USA. Currently working as scientist to improve bio-ethanol production and biomass utilization in an industrial setting.

Professional Positions:

Scientist in molecular biology department at Xyleco. November 27, 2017 till July 6, 2018. As a scientist I worked to improve bio-ethanol production and biomass utilization using biochemical and molecular biological tools in an industrial setting. I have experience in using 3 liter to 6000 liters fermenters. RESEARCH SKILLS:

• Single molecule microscopy – Single-molecule flow-stretching experiments in TIRF microscopes and DNA motion capture assay to investigate the molecular mechanisms like fully reconstituted replication products in single molecule microscopy and different polymerase interaction (Pol III vs Pol IV). Data analysis using ImageJ, MicrobeTracker etc.

• Biochemistry – In vitro DNA replication: biochemistry and single molecule analysis. DNA sequencing, primer extension reaction, EMSA and DNase I footprinting. Highly purified DNA

(damaged or undamaged DNA based templetes ). Lambda phage viral production and purification.

• Molecular biology – DNA/RNA Isolation, PCR, RT-PCR, qPCR, De novo gene synthesis, SOE/OE PCR, Cloning, Vector design/construction, Cell-line engineering, Site-directed mutagenesis, Southern hybridization/Gene copy number analysis etc.

• Protein expression – Bacterial and Yeast. Transient and stable transfections. Antibody generation in laboratory animals

• Protein Purification – Preparative/analytical filtration, separation, precipitation, chromatography etc. Affinity, IEX, HIC, RP etc. I have extensive experience in purifying various proteins using different columns and methods.

• Protein/Biochemical Characterization- PAGE, SDS-PAGE, Western Blot, ELISA, DLS, Analytical HPLC, Mass spectrometry (MALDI-TOF), 2-D, mass fingerprinting etc.

• Assay development/validation/screening – Mechanistic enzymology, Colorimetric, Fluorescence

(FRET, FP, Fluorescence Quenching), Radiometric etc. Enzyme kinetics, KM, Vmax, IC50, EC50 etc.

• Biophysical spectroscopy – Fluorescence, CD etc.

• Bioinformatics – BLAST, Clustal, Lasergene, Vector NTI etc.

• Microbiology – Handling of E. coli, M. smegmatis, and M. tuberculosis H37Ra cultures transformation, Growth/Kill curves, Antibiotic sensitivity, MIC, LD50 etc. EDUCATION: Postdoctoral research

Harvard Medical School, Boston. 2015-2017

• Force-based multiplexed single-molecule experiments (e.g. flow-stretching assay and DNA motion capture).

• Developing hybrid approaches to study how bacterial replisomes respond to damage in the leading strand.

• Single molecule assay to look into exchange and timing of Pol IV arrival and interaction(s) in replisomes during damage response.

• Over-expressed, purified and characterized several proteins/ enzymes assosiated with replication and damage response.

Memorial Sloan Kettering Cancer Center, New York. 2009-2015

• Isolated highly pure single stranded phage DNA using sucrose and cesium chloride gradients.

• Designed and generated a supercoiled DNA template which when replicated the moving fork encounters the damage in the leading-strand damage. We developed this template to examine the consequences of collision of the replisome with template damage.

• Using this bona fide stalled replication fork I investigated the replication fork regression activity of several proteins implicated to be involved in such process like RecA, RecG and RuvAB.

• I was successfully able to show that both RecG and RuvAB can regress the stalled fork in the presence of the replisome and SSB however, RuvAB generates a completely unwound product, whereas RuvC cleaves the Holliday junction that are generated by RecG-catalyzed fork regression.

• On the other hand if the replication proteins are removed from the stalled fork by protease digestion RecA catalyzes net regression only when the Okazaki fragments are sealed.

• Our experiments have also shown that RecG stimulates RuvAB-catalyzed regression whereas there is no such collaboration with RecA.

• We published these observations in two Journal of Biological Chemistry papers. Institute of Microbial Technology, India. Ph. D. 2003-2008

• During my Ph. D. I characterize two-component system (TCS), PhoP-PhoR from M. tuberculosis and examined interaction(s) between the response regulator PhoP and its cognate DNA target.

• I cloned, over-expressed, purified and characterized PhoP and PhoR proteins from Mtb H37Ra for the first time.

• I also for the first time standardized the autophosphorylation conditions for PhoR and transphosphorylation conditions for PhoP proteins and established that Mtb phoP and phoR gene products encode for a functional TCS; identifiying the primary sites of phosphorylation in both the proteins.

• I developed an in vivo assay to show that phoP is transcribed via autoregulation showing DNA binding properties of response regulator protein PhoP and the effect of phosphorylation of PhoP on DNA binding.

• I used footprinting assay to reveal three 9-bp direct repeat units with the consensus binding site.

• I determined Lys141-Arg247 as the functional C-terminal domain (PhoPc) that binds to the target DNA and my experiments suggested that the N-terminal domain is not merely a regulator of phosphorylation- dependent protein-protein interactions of PhoP protomers, but is also a modulator of DNA binding functions.

• We were also able to propose a head-to-head model that accounts for the DNA binding and protein- protein cross-linking data.

• We were able to publish these observations in series of papers (4) and the project is still continuing with the help of new students.

List of Research Publications

1. Gupta, S., Chakrabarti, P. and Sarkar, D. (2005) Nucleotide Induced Conformational Change in the Catalytic Subunit of the Phosphate Specific Transporter from M. tuberculosis: Implications for the ATPase Structure. Biochim. Biophys. Acta 1750, 112-21.

2. Gupta, S., Sinha, A. and Sarkar, D. (2006) Transcriptional autoregulation by Mycobacterium tuberculosis PhoP involves recognition of novel direct repeat sequences in the regulatory region of the promoter. FEBS Letters 580, 5328-5338.

3. *Sinha, A., *Gupta, S., Bhutani, S., Pathak, A. and Sarkar, D. (2007) PhoP-PhoP interaction on adjacent PhoP binding sites is influenced by protein phosphorylation. J. Bacteriol. 190, 1317-1328 (*contributed equally to the manuscript).

4. Gupta, S., Pathak, A. Sinha, A., and Sarkar, D. (2009) Mycobacterium tuberculosis PhoP recognizes two adjacent direct-repeat sequences to form a head-to-head dimers. J.Bacteriol. 191, 7466-7476. 5. Gupta, S., Yeeles J. T. P. and Marians, K. J. (2014) Regression of Replication Forks Stalled by Leading- strand Template Damage I. Both RecG and RuvAB Catalyze Regression, but RuvC Cleaves the Holliday Junctions Formed by RecG Preferentially. JBC. 289(41) 283**-*****. 6. Gupta, S., Yeeles J. T. P. and Marians, K. J. (2014) Regression of Replication Forks Stalled by Leading- strand Template Damage II. Regression by RecA Is Inhibited by SSB. JBC. 289 (41) 28388–28398. M. Sc. in Biotechnology. (2000-2002, 1st class)

V. B. S. Purvanchal University, Jaunpur, UP, India. B. Sc. in Biological Sciences (1996-1999, Chemistry, Botany and Zoology, 1st class) Ewing Christian College, Allahabad University, Allahabad, UP, India. AISSCE (Class X, 1993, 1st division) and AISSE (Class XII, 1995, 1st division) CBSE, New Delhi, India.

Currently I am interested in working in commercial and industrial setting to apply my skills and experience in DNA replication and protein purification. I think I can be an asset in such settings. If possible, I would like to visit and I would be happy to present my work. My references will be happy to provide letters of recommendations. I look forward to hearing from you and encourage you to contact me if you need more information.

Awards/Fellowships

Silver medallist, M. Sc. Purvanchal University

CSIR-UGC NET qualified in Dec 2003 as Junior Research Fellow GATE (Graduate Aptitude Test for Engineers) qualified in 2002 Passed Certificate in Computing in 2000 from IGNOU Symposiums attended and poster presented:

1) Presented a poster entitled “Insights into the mechanism of gene regulation by the Mycobacterium tuberculosis response regulator PhoP” at the International Symposium on Chemical Biology held at Indian Institute of Chemical Biology, Kolkata. India from March 7-9, 2007. 2) Presented a poster entitled “Transcription regulation by Mycobacterium tuberculosis PhoP: PhoP- PhoP interaction at adjacent PhoP binding sites is influenced by protein phosphorylation” at the Tenth Asian Conference on Transcription (ACT-X) held at Indian Institute of Science, Bangalore, India from January 13-16, 2008.

3) Attended 2nd Chandigarh Science Congress, CHASCON-2008 held at Punjab University, Chandigarh, India from March 14-15, 2008.

4) Presented a poster entitled “Role of RecG helicase from Escherichia coli at stalled forks.” at the FASEB Summer Research Conference (SRC) held in Steamboat, Colorado, USA from 5th Aug to 10th Aug 2011.

References

1. Prof. Kenneth J. Marians 3. Joseph J. Loparo, Ph.D. Molecular Biology Program, Department of Biological Chemistry Memorial Sloan Kettering Cancer Center, and Molecular Pharmacology New York, NY 10065 Harvard Medical School (HMS)

********@**************.***

Phone : 917-***-****

250 Longwood Avenue

2. Dr. Dibyendu Sarkar Boston, MA 02115, USA

Scientist, Institute of Microbial Technology Email: *************@***.*******.*** Sector-39A, Chandigarh – 160036 India. Phone: +1-617-***-**** Email: ********@******.***.**

Phone +91-987*******

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