Zahid Alim Ansari

Title: Investigating the therapeutic potential of Piperlongumine via targeting differentially expressed genes through integrative computational and in vitro validation in colorectal cancer.

• Conducted systematic retrieval and preprocessing of colorectal cancer gene expression datasets, followed by differential gene expression (DEG) analysis using R and Bioconductor packages.
• Performed functional annotation and pathway enrichment analysis (GO, KEGG) to identify molecular pathways altered in colorectal cancer.
• Constructed protein–protein interaction (PPI) networks using STRING and analysed molecular clusters and hub genes using Cytoscape/MCODE.
• Screened Piperlongumine (PIP) as a therapeutic candidate by integrating in silico docking, binding energy evaluation, and pharmacokinetic/ADMET profiling.
• Validated target genes experimentally through cell and molecular assays, including RNA extraction, cDNA preparation, PCR, relative expression analysis, and Western blotting where applicable.
• Designed a comprehensive workflow combining transcriptomics, network biology, molecular docking, and wet-lab validation.
• Performed statistical analysis, curated datasets, maintained lab documentation, and ensured reproducibility of all computational and laboratory protocols.
• Interpreted results, prepared figures, drafted the manuscript, and contributed to revisions leading to acceptance in Pathology – Research and Practice (Elsevier).

Title: Cloning of guide DNA in px459 vector for downstream knock-in of Histone H4 (H4-16) with fluorescence marker.

• Performed plasmid DNA isolation using the PureLink HiPure Maxiprep protocol, including cell harvesting, alkaline lysis, column purification, ethanol precipitation, and resuspension.
• Prepared LB broth and Luria Agar plates for bacterial culture maintenance, transformation, and antibiotic selection.
• Designed guide DNA (gDNA) sequences targeting the Histone H4 (H4C16) gene using genome-editing design tools and validated off-target/pam compatibility.
• Linearized the px459 v2.0 CRISPR-Cas9 plasmid using BbsI restriction digestion and verified digestion by agarose gel electrophoresis.
• Conducted guide oligo annealing using T4 ligase buffer and T4 PNK followed by controlled thermal cycling for duplex formation.
• Performed ligation of annealed gDNA into linearized px459 plasmid, optimizing ligation conditions and incubating reactions overnight.
• Prepared competent E. coli (STBL-3) cells via CaCl₂ method and performed transformation using the heat-shock protocol.
• Plated transformed cells on antibiotic selection plates, carried out replica plating, and isolated single colonies for screening.
• Conducted colony PCR using Px459 forward primer and guide-specific reverse primers to confirm positive clones.
• Analysed 2% agarose gel electrophoresis results to identify correct insertion (~270 bp band) and validate successful cloning.
• Prepared glycerol stocks of successfully cloned constructs for long-term storage and future downstream applications.

• Microbial culturing, isolation, enumeration, preservation, and aseptic techniques
• Staining methods: Gram, spore, capsule, flagella, negative and differential staining
• Biochemical identification of microbes (IMViC tests, enzyme assays: amylase, catalase, urease, phosphatase, etc.)
• Antibiotic susceptibility testing and MIC determination
• Growth kinetics: growth curve preparation, turbidimetry, cell count, spectrophotometry
• Fermentation techniques and microbial product assays
• Water, air, soil, and food microbiological analysis (MPN, microbial load tests, spoilage detection)
• Plant pathology: pathogen isolation, disease diagnostics, host–pathogen interaction studies
• Analytical techniques: agarose & PAGE electrophoresis, paper & TLC chromatography, Km and Vmax determination
• Molecular biology: plasmid isolation, restriction digestion, PCR, primer design, bacterial transformation, DNA electrophoresis
• Immunology techniques: ELISA, immunodiffusion, immunoelectrophoresis, blood grouping, antigen–antibody reactions