From f4b6908e40f10ea285b3a9bbdd89fe15b68c3bed Mon Sep 17 00:00:00 2001 From: ChaceJerry <30697733+sunmichace@users.noreply.github.com> Date: Wed, 11 Oct 2023 10:51:24 +0800 Subject: [PATCH] Update protocols.md --- docs/documentation/protocols.md | 262 ++++++++++++++++++++++++++++++++ 1 file changed, 262 insertions(+) diff --git a/docs/documentation/protocols.md b/docs/documentation/protocols.md index ed14236..b97a135 100755 --- a/docs/documentation/protocols.md +++ b/docs/documentation/protocols.md @@ -1,2 +1,264 @@ # 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. + +