Update protocols.md

docs/documentation/protocols.md

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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.
+
+