Notebook

Lab Notebooks

This page contains all of our detailed lab notebooks documenting our experimental procedures, results, and analysis throughout the project. Each notebook covers different aspects of our research including characterization, construct development, transcriptomics analysis, and protein purification.

Notebook	Description	Download
Characterization Notebook	Comprehensive biophysical characterization experiments including MST binding assays, enzymatic activity assays, CD spectroscopy, and AUC sedimentation velocity analysis for TYMS-GFP protein.
Construct Notebook	Detailed documentation of construct design, cloning procedures, transformation protocols, and validation experiments for all protein constructs used in the project.
Transcriptomics Notebook	Complete transcriptome analysis workflow documenting E. coli BL21 response to PFOA exposure, including RNA extraction, library preparation, and sequencing protocols.
TYMS-GFP Purification Notebook	Detailed protocol for expressing and purifying His-tagged TYMS-GFP fusion protein in E. coli, including large-scale culture, induction, and purification procedures.
TYMS Purification Notebook	Automated purification protocol for non-tagged TYMS protein using Bio-Rad Profinia system, including high-throughput production and quality control procedures.

Characterization Notebook

This notebook documents our comprehensive biophysical characterization experiments for the TYMS-GFP protein. The experiments include buffer preparation, protein handling, and advanced analytical techniques to study protein structure, function, and interactions.

Key Experiments Documented:

• Buffer Preparation: Tris-NaCl-TCEP buffer preparation, matched phosphate buffers for CD/AUC analysis, and TES buffers with formaldehyde fixative
• Protein Buffer Exchange: Spin desalting protocols and concentration measurements using Beer-Lambert Law
• Fluorescent Labeling: Covalent labeling with Sulfo-Cyanine5 NHS ester and degree of labeling (DOL) calculations
• MST Binding Assays: Serial dilution protocols, binding affinity (Kd) determination, and signal-to-noise optimization
• Enzymatic Activity Assays: TYMS activity monitoring, substrate kinetics, and spectrophotometric analysis
• CD Spectroscopy: Secondary structure analysis and mean residue ellipticity (MRE) calculations
• Analytical Ultracentrifugation: Sedimentation velocity analysis, oligomeric state determination, and hydrodynamic characterization

Key Results:

• PFOA Binding: MST experiments confirmed binding between TYMS-GFP and PFOA with Kd values of 166 mM and 109 µM
• Positive Controls: dUMP binding showed high affinity (Kd = 178 nM) confirming expected strong binding to TYMS active site
• Negative Controls: mTHF showed no significant binding, as expected
• Ternary Complex: Labeled TYMS bound strongly to dUMP/mTHF substrate mixture (Kd = 1.93 µM)
• Secondary Structure: CD analysis confirmed proper protein folding and secondary structure
• Oligomeric State: AUC analysis determined protein oligomeric state and homogeneity

Construct Notebook

This notebook contains detailed documentation of all construct design, cloning procedures, and validation experiments. It covers the development of various protein constructs used throughout the project.

Constructs Documented:

• TYMS-GFP Fusion: His-tagged fusion protein for fluorescence-based assays
• TYMS Protein: Non-tagged TYMS for enzymatic and binding studies
• Control Constructs: Various control proteins for validation experiments

Key Procedures:

• Design Phase: Construct design with appropriate linkers and tags
• Cloning: PCR amplification, restriction digestion, and ligation procedures
• Transformation: E. coli transformation and selection protocols
• Validation: Colony PCR, restriction digest, and sequencing verification
• Expression Testing: Small-scale expression trials and optimization

Transcriptomics Notebook

This comprehensive notebook documents the complete transcriptome analysis workflow for studying E. coli BL21 response to PFOA exposure. The experiments span from bacterial culture and exposure through RNA extraction, library preparation, and sequencing.

Experimental Design:

• Timepoints: 1 hour and 4 hour exposure periods
• Concentrations: 100 µM, 1 µM, 0.01 µM, and untreated controls
• Replicates: 3 biological replicates per condition
• Total Samples: 24 samples (2 timepoints × 4 concentrations × 3 replicates)

Key Procedures:

• Bacterial Culture: E. coli BL21 overnight culture and experimental setup
• PFOA Exposure: Serial dilution preparation and exposure protocols
• RNA Extraction: Commercial kit with on-column DNase treatment and lysozyme digestion
• Quality Control: Spectrophotometry (Nanodrop), fluorometry (Qubit), and integrity analysis (Bioanalyzer)
• Library Preparation: rRNA depletion, cDNA synthesis, fragmentation, adaptor ligation, and PCR enrichment
• Final QC: Bioanalyzer analysis for library size distribution and molar concentration

Key Results:

• RNA Quality: High-quality RNA with A260/280 ratios ~2.0 and RIN values of 9-10
• Library Quality: Successful library preparation with appropriate size distributions
• Sequencing Ready: Libraries prepared for Illumina sequencing to analyze differential gene expression

TYMS-GFP Purification Notebook

This notebook documents the complete process for expressing and purifying His-tagged TYMS-GFP protein in E. coli. The protocol covers large-scale culture through final protein concentration and analysis.

Protocol Overview:

• Part 1 - Protein Expression (Days 1-3): Large-scale culture growth, protein induction, and cell harvesting
• Part 2 - Protein Purification (Days 4-6): Cell lysis, affinity chromatography, size exclusion chromatography, and final concentration

Key Procedures:

• Large-Scale Culture: 4-liter E. coli BL21 cultures with ampicillin selection
• Protein Induction: IPTG induction at OD600 0.6-0.8, followed by overnight expression at 18°C
• Cell Harvesting: Centrifugation at 9000 x g for 12 minutes at 4°C
• Cell Lysis: Sonication with DNase1 and PMSF treatment
• Nickel Affinity Chromatography: His-tag purification using gravity flow columns
• Size Exclusion Chromatography: Final purification using HiLoad 16/600 Superdex 75 pg column
• Quality Control: SDS-PAGE analysis and GFP fluorescence verification

Expected Results:

• Protein Yield: Purified TYMS-GFP protein with GFP fluorescence verification
• Purity: SDS-PAGE analysis showing clear protein bands with high purity in final elution fractions
• Fluorescence: Characteristic GFP fluorescence when visualized under appropriate lighting conditions

TYMS Purification Notebook

This notebook details the automated expression and purification of non-tagged TYMS protein using the Bio-Rad Profinia Protein Purification System for high-throughput protein production.

Protocol Overview:

• Part 1 - Protein Expression (Day 1-2): Overnight culture inoculation, large-scale growth, IPTG induction, and cell harvesting
• Part 2 - Automated Purification (Day 2): Cell lysis, automated IMAC purification, desalting, and protein collection

Key Procedures:

• Large-Scale Culture: 4-liter E. coli BL21 cultures with ampicillin selection and overnight inoculation
• Protein Induction: IPTG induction at OD600 >1.0, followed by 3-hour expression at 37°C
• Cell Harvesting: Centrifugation at 9,000 x g for 12 minutes at 4°C
• Automated Lysis: RIPA buffer resuspension with protease inhibitors and sonication
• Automated IMAC Purification: Bio-Rad Profinia system with nickel-charged IMAC cartridge
• Automated Desalting: Bio-Gel P-6DG desalting cartridge for buffer exchange
• Quality Control: SDS-PAGE analysis and spectrophotometric concentration determination

Expected Results:

• High Purity: Purified, non-tagged TYMS protein with high purity and concentration
• Consistent Quality: Automated fraction collection ensuring consistent protein quality
• Ready for Use: Automated buffer exchange and desalting for immediate use in downstream applications

Notebook Summary

These lab notebooks provide comprehensive documentation of our experimental work throughout the project. They demonstrate our systematic approach to protein characterization, construct development, transcriptomics analysis, and protein purification. Each notebook contains detailed protocols, results, and analysis that support our overall research objectives.

The notebooks show our commitment to rigorous experimental design, proper documentation, and quality control throughout all aspects of our research. They serve as a complete record of our experimental work and provide valuable resources for future research and collaboration.