Spring Lab Work: Retrying Our Constructs
This protocol outlines our spring laboratory work focused on retrying and optimizing our construct designs. The experiments were designed to validate and improve upon our previous construct designs through systematic testing and optimization.
| Protocol | Description | Download |
|---|---|---|
| Spring Construct Protocol | Comprehensive protocol for retrying our constructs including transformation, selection, mini-prep, restriction digest, gel electrophoresis, screening, and storage procedures. |
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Protocol Overview
Our spring construct protocol encompasses a comprehensive 5-day laboratory schedule designed to systematically retry and validate our construct designs. The protocol includes:
- Day 1 (Monday): Pour plates and transformations to BL21
- Day 2 (Tuesday): Selection of colonies and incubation
- Day 3 (Wednesday): Mini-prep, digest, gel electrophoresis, and incubation of good colonies
- Day 4 (Thursday): PFAS exposure/screening day with overnight incubation and storage stock creation
- Day 5 (Friday): Results evaluation and lab notebook digitization
Key Experimental Components
The protocol includes detailed procedures for:
- Plate Preparation: LB-agar plates with appropriate antibiotic selection (Kanamycin and Ampicillin)
- Transformation: E. coli BL21 competent cell transformation with multiple constructs
- Selection and Culture: Colony selection and overnight culture preparation
- DNA Extraction: Mini-prep protocol using GeneJET kit for plasmid purification
- Verification: Restriction digest and gel electrophoresis for construct validation
- Screening: PFAS exposure experiments with detailed calculation protocols
- Storage: Glycerol stock preparation for long-term storage
Materials and Equipment
Key materials include autoclave, fume hood, selective agar plates, competent cells, antibiotics, molecular biology kits, and specialized equipment for gel electrophoresis and screening assays.
Expected Results
The protocol is designed to yield specific band patterns on gel electrophoresis corresponding to different construct sizes, with detailed expectations for each construct type including pRMA (1057 bp), Fab-GFP (1329 bp), and Estradiol constructs with specific fragment sizes.
TYMS-GFP Protein Production Protocol
This comprehensive protocol outlines the complete process for expressing and purifying His-tagged TYMS-GFP protein in E. coli, from large-scale culture through final protein concentration and analysis.
| Protocol | Description | Download |
|---|---|---|
| TYMS-GFP Protein Production | Complete protocol for expressing and purifying His-tagged TYMS-GFP protein in E. coli, including large-scale culture, induction, cell lysis, nickel affinity chromatography, and size exclusion chromatography. |
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Protocol Overview
The TYMS-GFP protein production protocol is a 6-day process divided into two main phases:
- 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 Experimental Components
The protocol includes detailed procedures for:
- 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
Materials and Equipment
Key materials include LB broth, ampicillin, IPTG, lysis buffer (50 mM Tris, 500 mM NaCl, 30 mM Imidazole), elution buffer (50 mM Tris, 500 mM NaCl, 300 mM Imidazole, 5 mM BME), nickel beads, SEC buffer (50 mM Tris, 100 mM NaCl, 5 mM TCEP), and specialized equipment for sonication, centrifugation, chromatography, and protein analysis.
Expected Results
The protocol is designed to yield purified TYMS-GFP protein with GFP fluorescence verification. SDS-PAGE analysis should show clear protein bands corresponding to the expected molecular weight, with high purity in the final elution fractions. The protein should exhibit characteristic GFP fluorescence when visualized under appropriate lighting conditions.
Automated TYMS Production Protocol
This protocol 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 | Description | Download |
|---|---|---|
| Automated TYMS Production | Automated protein expression and purification protocol for non-tagged TYMS protein using Bio-Rad Profinia system, including large-scale culture, induction, automated IMAC purification, and desalting. |
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Protocol Overview
The automated TYMS production protocol is a streamlined 2-day process designed for high-throughput protein production using the Bio-Rad Profinia Protein Purification System:
- 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 Experimental Components
The protocol includes detailed procedures for:
- 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
Materials and Equipment
Key materials include LB media, ampicillin, IPTG, RIPA buffer, protease inhibitor cocktail, β-mercaptoethanol, binding buffer (50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole), elution buffer (50 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole), storage buffer (PBS), and the Bio-Rad Profinia Protein Purification System with IMAC and desalting cartridges.
Expected Results
The automated protocol is designed to yield purified, non-tagged TYMS protein with high purity and concentration. SDS-PAGE analysis should show clear protein bands corresponding to the expected molecular weight, with automated fraction collection ensuring consistent protein quality. The system provides automated buffer exchange and desalting for immediate use in downstream applications.
Biophysical Characterization Protocols
This comprehensive collection of protocols covers advanced biophysical characterization techniques including buffer preparation, protein handling, and analytical assays for studying protein structure, function, and interactions.
| Protocol | Description | Download |
|---|---|---|
| Biophysical Characterization Protocols | Complete collection of protocols for buffer preparation, protein handling, and analytical characterization including MST binding assays, enzymatic activity assays, CD spectroscopy, AUC sedimentation velocity, and fluorescent labeling procedures. |
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Protocol Overview
The biophysical characterization protocols are organized into three main sections covering comprehensive protein analysis workflows:
- Section 1 - Buffer Preparation: Tris-NaCl-TCEP buffer, matched phosphate buffers for CD/AUC, TES buffers with and without formaldehyde
- Section 2 - Protein Handling: Buffer exchange protocols, fluorescent labeling procedures for MST experiments
- Section 3 - Analytical Assays: MST binding assays, enzymatic activity assays, CD spectroscopy, and AUC sedimentation velocity
Key Experimental Components
The protocols include detailed procedures for:
- Buffer Preparation: Tris-NaCl-TCEP buffer (1L), matched phosphate buffers for CD/AUC analysis, TES buffers with formaldehyde fixative
- Protein Buffer Exchange: Spin desalting columns for buffer exchange, concentration measurement using Beer-Lambert Law
- Fluorescent Labeling: Covalent labeling with Sulfo-Cyanine5 NHS ester, degree of labeling (DOL) calculation
- MST Binding Assays: Serial dilution protocols, binding affinity (Kd) determination, signal-to-noise optimization
- Enzymatic Activity Assays: TYMS activity monitoring, substrate kinetics, spectrophotometric analysis
- CD Spectroscopy: Secondary structure analysis, mean residue ellipticity (MRE) calculations
- Analytical Ultracentrifugation: Sedimentation velocity analysis, oligomeric state determination, hydrodynamic characterization
Materials and Equipment
Key materials include Tris base, NaCl, TCEP, EDTA, β-mercaptoethanol, formaldehyde, Sulfo-Cyanine5 NHS ester, dUMP, mTHF substrates, Zeba spin desalting columns, and specialized equipment including spectrophotometers, CD spectrometers, AUC instruments, and MST devices.
Expected Results
The characterization protocols are designed to provide comprehensive protein analysis including binding affinities (Kd values), enzymatic activity profiles, secondary structure confirmation, oligomeric state determination, and hydrodynamic properties. These techniques enable detailed understanding of protein structure-function relationships and interaction mechanisms.
Transcriptomics Analysis Protocol
This comprehensive protocol outlines the complete transcriptome analysis workflow for studying E. coli BL21 response to PFOA exposure, from bacterial culture and exposure through RNA extraction, library preparation, and sequencing.
| Protocol | Description | Download |
|---|---|---|
| Transcriptomics Analysis Protocol | Complete transcriptome analysis protocol for E. coli BL21 response to PFOA exposure including bacterial culture, RNA extraction, library preparation, and quality control procedures. |
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Protocol Overview
The transcriptomics analysis protocol is a comprehensive 4-part workflow designed to analyze gene expression changes in E. coli BL21 in response to PFOA exposure:
- Part 1 - Bacterial Culture and PFOA Exposure: E. coli BL21 culture setup, PFOA exposure at varying concentrations, and cell harvesting
- Part 2 - RNA Extraction and Quality Control: Total RNA isolation, DNase treatment, and quality assessment using spectrophotometry and bioanalyzer
- Part 3 - RNA-Seq Library Preparation: rRNA depletion, cDNA synthesis, library preparation with barcoding for Illumina sequencing
- Part 4 - Final Library Quantification: Quality control and concentration determination for sequencing
Key Experimental Components
The protocol includes detailed procedures for:
- Bacterial Culture Setup: E. coli BL21 overnight culture, experimental culture inoculation, and PFOA exposure at concentrations ranging from 0.01 µM to 100 µM
- PFOA Exposure: Serial dilution preparation, exposure at multiple timepoints (1 hour and 4 hours), and cell harvesting at specific OD600 values
- RNA Extraction: Commercial RNA extraction kit with on-column DNase treatment, lysozyme digestion, and multiple purification steps
- Quality Control: Spectrophotometry (Nanodrop), fluorometry (Qubit), and integrity analysis (Bioanalyzer) with RIN assessment
- Library Preparation: rRNA depletion using NEBNext kit, cDNA synthesis, fragmentation, adaptor ligation, and PCR enrichment
- Final QC: Bioanalyzer analysis for library size distribution and molar concentration determination
Materials and Equipment
Key materials include E. coli BL21 frozen stock, LB broth, PFOA stock solution (20 mM), RNA extraction kits, NEBNext rRNA Depletion Kit, NEBNext Ultra II Directional RNA Library Prep Kit, and specialized equipment including spectrophotometers, thermal cyclers, centrifuges, and bioanalyzers.
Expected Results
The protocol is designed to yield high-quality RNA-seq libraries suitable for Illumina sequencing. Quality control metrics should show A260/280 ratios of ~2.0, A260/230 ratios of 2.0-2.2, RIN values of 9-10, and library size distributions with distinct peaks in the 200-500 bp range with minimal adaptor-dimer contamination. The final libraries should be ready for equimolar pooling and sequencing to analyze differential gene expression in response to PFOA exposure.