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Protocols

1. Processing of RNA-Seq Data

Materials: - Computer with installed R studio and relevant bioinformatics packages. - Human tissue RNA-seq data from specified databases.

Procedure: 1. Preparation: Install necessary packages and libraries on R studio. Ensure stable internet connectivity for data download. 2. Data Download: - Navigate to the respective database web pages. - Select the desired datasets and initiate download. - Store data in a specified folder for easy access. 3. Processing: - Load the data into R studio. - Use the DESeq2 package (or similar) for differential expression analysis. - Filter out genes with low counts. - Normalize the data. 4. Analysis: - Generate heatmaps, PCA plots, and other relevant plots to visualize gene expression patterns. - Save and catalog results.

Specific Conditions: - Ensure the computer has sufficient memory and processing power for large datasets.

2. Protein Tertiary Structure Prediction and Mutation Analysis

Materials: - Computer with internet access. - AlphaFold2 platform access. - UCSF Chimera software. - DNA sequences for the target proteins.

Procedure: 1. Protein Prediction: - Navigate to the Bkunyun Platform and upload the DNA/protein sequence. - Initiate AlphaFold2 prediction and wait for results. 2. Structural Analysis: - Download the resulting PDB files. - Open the PDB files in UCSF Chimera. - Visualize, compare, and analyze structures. 3. Mutation Analysis: - Highlight and label mutation sites on the structures using UCSF Chimera tools. - Predict potential functional changes due to mutations.

Specific Conditions: - Ensure a stable internet connection for online platforms. - Ensure sufficient storage space for saving PDB and other files.

3. Collagen and Elastin Purification

a. Chromatography-free purification method

Materials: - BL21 (DE3) strain with appropriate plasmid. - PBS (pH 9.0, 0.5 M NaCl). - 10 KDa & 1 KDa ultrafiltration tubes. - HPLC system.

Procedure: 1. Cultivation: - Inoculate a fresh culture of BL21 in LB medium (pH 8.5) supplemented with 50 mg/L kanamycin. - Incubate at 37°C, 200 rpm until OD600 ~0.6. - Add 0.3 mM IPTG to induce protein expression and incubate overnight. 2. Protein Extraction: - Harvest cells by centrifugation at 4,000 rpm, 15 minutes, 4°C. - Resuspend the cell pellet in 50 mL PBS. - Lyse cells using high-pressure homogenization. - Centrifuge lysed cells at 10,000 rpm, 30 minutes, 4°C to remove cell debris. 3. Ultrafiltration: - Pass supernatant through a 10 KDa ultrafiltration tube to remove proteins >10 KDa. - Adjust pH of filtrate to 6.5 using 1 M HCl. - Incubate at 30°C overnight for potential peptide cleavage. - Pass the solution through a 1 KDa ultrafiltration tube. 4. Quality Control: - Analyze filtrate using HPLC to assess peptide purity and concentration.

Specific Conditions: - All steps involving cells and proteins should be done on ice or at 4°C, unless otherwise stated. - Use sterile techniques to prevent contamination.

4. ARTP-PANCE for Screening Intein and T7 RNAP Mutants In Vivo

Materials: - Primary M13 phage for GyrA or T7 RNAP. - S1030 strain with pJC175e plasmid. - DNA-LAMP method kit. - LB medium, appropriate antibiotics.

Procedure: 1. Phage Addition: - Inoculate a fresh culture of S1030 strain containing the pJC175e plasmid. - At early log phase, add the primary M13 phage. - Incubate to allow for bacterial-phage interactions. 2. ARTP Mutation: - Centrifuge and re-suspend bacterial cells in fresh LB medium. - Expose to ARTP treatment in controlled settings for specified durations (e.g., 30s, 120W). - Dilute treated cells in fresh LB medium. 3. Phage Titer Estimation: - Use the DNA-LAMP method to periodically measure phage titers. - When phage titer reaches 10^6 CFU/mL, proceed to the next step. 4. Phage Collection: - Centrifuge culture and collect the supernatant containing the M13 phage.

Specific Conditions: - Use sterile conditions throughout to prevent contamination. - The ARTP mutagenesis equipment should be calibrated to provide the right exposure intensity. - Monitor cultures for any signs of contamination or cell death.

5. CRISPR-Associated Transposon Protocol

Materials: - BL21 strain containing specified plasmids. - LB medium with appropriate antibiotics. - PCR equipment, reagents, and primers. - Sanger sequencing setup.

Procedure: 1. Strain Induction: - Inoculate BL21 strain with the necessary plasmids in LB medium with required antibiotics. - Grow until OD600 reaches 0.6. - Induce with 0.2 mM IPTG and continue incubation overnight. 2. Isolation: - Plate culture on LB plates with antibiotics to isolate monoclonal strains. - Grow plates until colonies are visible. 3. Identification: - Use bacterial PCR to identify strains with the desired insert fragments. - Confirm insertions through Sanger sequencing.

Specific Conditions: - Use a controlled incubator set at 37°C for bacterial growth. - Maintain sterility throughout to avoid contamination. - Optimize PCR conditions for specific primers and target regions.

6. TADR-FADS for Screening Proline Hydroxylase Mutants

Materials: - BL21 strain with specific genomic modifications and plasmids. - Fluorescent microplate reader. - LB medium and appropriate antibiotics.

Procedure: 1. Strain Transformation: - Transform BL21 with the desired plasmid. - Grow transformed cells in LB medium with appropriate antibiotics until OD600 ~0.6. 2. Induction: - Add 0.5 mM IPTG to the culture. - Continue overnight incubation. 3. Fluorescence Measurement: - Measure GFP fluorescence intensity of the cultures using a fluorescent microplate reader.

Specific Conditions: - Maintain cultures at 37°C, 200 rpm throughout. - Calibration of the microplate reader is essential for accurate fluorescence readings.

7. Microfluid Chip and Device Preparation

Materials: - PDMS - HFE7500 fluorinated oil. - Microscopy and imaging setup with lasers and cameras.

Procedure: 1. Chip Fabrication: - Create microfluidic patterns using PDMS. - Allow PDMS to cross-link and cure. - Punch the necessary ports and channels. 2. Assembly: - Attach the PDMS structure to a glass slide. - Apply a hydrophobic surface coating. - Fill electrode holes as necessary. 3. Droplet Generation: - Use a dropmaker chip with fluorinated oil to generate droplets. - Employ imaging equipment to monitor and manipulate droplets.

Specific Conditions: - PDMS curing should be done under controlled temperature conditions for consistency. - Maintain a clean environment to ensure dust or contaminants do not interfere with microfluidic operations.

8. Activity Assay for Proline Hydroxylase

Materials: - Bacterial Protein Extraction Kit. - Hydroxyproline (HYP) content detection kit.

Procedure: 1. Protein Extraction: - Extract total proteins from E. coli using the extraction kit. 2. Quantification: - Measure protein concentration using a BCA protein quantification kit. 3. Activity Assessment: - Determine proline hydroxylation levels using the HYP content detection kit.

Specific Conditions: - All extraction steps should be carried out at 4°C or on ice to maintain protein stability. - Ensure that all reagents, especially those in the HYP detection kit, are at the appropriate temperature and freshly prepared. - Control samples (non-hydroxylated proline samples) should be run alongside experimental samples for accurate comparison.

9. Collagen and Elastin Synthesis via CFPS Reaction

Materials: - 20 standard amino acids, glycine, proline, 2-oxoglutarate, etc. - Cell extract from a high yield source. - Target plasmid or DNA encoding collagen/elastin sequences. - Necessary buffers and cofactors for in vitro transcription and translation.

Procedure: 1. Preparation: - Thaw all reagents on ice. - Prepare a master mix containing all the components except the cell extract and DNA. 2. Reaction Setup: - In a sterile reaction tube, mix the master mix, cell extract, and target DNA/plasmid. - Ensure thorough mixing without introducing bubbles. 3. Incubation: - Place the reaction tube in a controlled incubator set at 37°C. - Allow the reaction to proceed for 8 hours. 4. Analysis: - After incubation, analyze the reaction mixture for synthesized collagen/elastin using SDS-PAGE or Western Blot.

Specific Conditions: - Ensure all components are fresh and uncontaminated. This is crucial for the efficiency of the CFPS system. - Maintain a sterile environment during the setup to prevent external protein or RNA contamination. - Optimal temperatures for the CFPS system might vary depending on the specific cell extract source. Adjust accordingly.

10. Proline Hydroxylase Mutant Screening via Microfluidic Chips

Materials: - Microfluidic chips prepared from PDMS. - HFE7500 fluorinated oil. - Culture of strains containing the P4Hc mutant library. - Necessary buffers and reagents for droplet generation.

Procedure: 1. Strain Preparation: - Grow the strains containing the P4Hc mutant library in LB medium until OD600 reaches ~0.6. - Dilute the culture for use in droplet generation. 2. Droplet Generation: - Set up the microfluidic chip on the appropriate device. - Introduce the bacterial culture and HFE7500 fluorinated oil into the chip to generate droplets. 3. Droplet Analysis: - Using the attached imaging system, monitor the droplets for desired phenotypes or activities. - Sort and collect droplets of interest for further analysis.

Specific Conditions: - Ensure consistent flow rates for both the bacterial culture and the fluorinated oil to generate uniform droplets. - Calibrate the imaging system for accurate detection and analysis of droplets. - Maintain a clean setup to prevent clogging or contamination within the microfluidic channels.