From 269816732c01fb35c2394409e44fd644a3e98571 Mon Sep 17 00:00:00 2001
From: Natalia <nataliavazpur@gmail.com>
Date: Tue, 1 Oct 2024 22:30:37 +0000
Subject: [PATCH] protocol lists
---
wiki/pages/experiments.html | 199 +++++++++++++++++++++++++++++++++---
1 file changed, 182 insertions(+), 17 deletions(-)
diff --git a/wiki/pages/experiments.html b/wiki/pages/experiments.html
index cd27301..88847fc 100644
--- a/wiki/pages/experiments.html
+++ b/wiki/pages/experiments.html
@@ -88,8 +88,8 @@
<p>All samples were imaged using fluorescent scanning confocal microscopy (Nikon confocal A1R, excitation 488-nm@16mW, emission filter 525/50) to evaluate fusion events. Cells were segmented using the transmitted light channel and instances of bright fluorescent spots within cells were evaluated. Image analysis was performed in ImageJ.</p>
</div>
- <!-- 8 -->
- <div class="h" id="eight">
+ <!-- 8 -->
+ <div class="h" id="eight">
<div class="h1">Protocols</div>
<div class="h2">Yeast Transformation</div>
<div class="h3">Materials and Reagents</div>
@@ -187,32 +187,197 @@
<li>Nuclease-free water to 25μl</li>
</ul>
</ol>
+ <div class="h2"><i>C. reinhardtii Transformation</i> </div>
+ <div class="h3">Materials and Reagents</div>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>C. reinhardtii cells (wall-less or walled cells treated with autolysin)</li>
+ <li>TAP medium</li>
+ <li>Ice</li>
+ <li>Sucrose</li>
+ <li>Transforming DNA</li>
+ <li>Salmon sperm carrier DNA (optional)</li>
+ <li>Water bath</li>
+ <li>20% sterilized corn starch suspension</li>
+ <li>PEG 8000</li>
+ <li>Agar plates with TAP medium and selective agent</li>
+ </ul>
+ <div class="h3">Equipment</div>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;"></ul>
+ <li>Gyratory shaker</li>
+ <li>Centrifuge</li>
+ <li>BioRad electroporation cuvette (4 mm gap)</li>
+ <li>BioRad Gene Pulser Xcell electroporator</li>
+ <li>Incubator tent with light</li>
+ </ul>
+ <div class="h3">Procedure</div>
+ <ol style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;"></ul>
+ <li><strong>Cell Culture</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>Grow cells in TAP medium at 27°C under light (50 μm photons/m² sâ»Â¹) on a gyratory shaker at 120 rpm until late exponential phase.</li>
+ <li>Chill cells on ice and harvest by centrifugation (880 × g for 5 min).</li>
+ <li>Resuspend cells in TAP + 40 mM sucrose at (1-4) × 10ⷠcells/mL.</li>
+ </ul>
+ </li>
+ <li><strong>Electroporation</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>Place 250 μL of concentrated cells at 16°C in the electroporation cuvette.</li>
+ <li>Add 2.5 μg transforming DNA and 50 μg salmon sperm carrier DNA (optional) to the cuvette.</li>
+ <li>Place cuvette in the electroporation device set at 1800-2300 nV/cm with capacitance at 10 μF and no shunt resistor.</li>
+ <li>Perform electroporation (time constant should be 5-6 ms).</li>
+ </ul>
+ </li>
+ <li><strong>Post-electroporation</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>Place the cuvette containing electroporated cells in a 25°C water bath for 5-30 min for recovery.</li>
+ </ul>
+ </li>
+ <li><strong>Plating</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>Prepare a suspension of 1 mL 20% sterilized corn starch in TAP + 40 mM sucrose + 0.4% PEG 8000.</li>
+ <li>Add an aliquot (0.4%-2% of sample) of electroporated cells to the suspension.</li>
+ <li>Spread the mixture onto plates containing TAP medium in 0.5% agar + agent for selective growth of transformants.</li>
+ </ul>
+ </li>
+ <li><strong>Incubation</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>Incubate plates under light (80 μm photons/m² sâ»Â¹) at 27°C until colonies are large enough for analysis.</li>
+ </ul>
+ </li>
+ </ol>
+ <p> Note: The expected transformation rate is approximately 10ⵠtransformants/μg DNA. </p>
<div class="h2">Yeast-<i>E. coli</i> Fusion</div>
<div class="h3">Materials and Reagents</div>
<ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
- <li>C. reinhardtii cells (wall-less or walled cells treated with autolysin)</li>
- <li>TAP medium</li>
- <li>Ice</li>
- <li>Sucrose</li>
- <li>Transforming DNA</li>
- <li>Salmon sperm carrier DNA (optional)</li>
- <li>Water bath</li>
- <li>20% sterilized corn starch suspension</li>
- <li>PEG 8000</li>
- <li>Agar plates with TAP medium and selective agent</li>
- </ul>
-
+ <li><strong>Strains</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li><strong>Escherichia coli:</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>NCM 3722 prototrophic K-12 strain with PlsB-msGFP2 integrated into the chromosome</li>
+ <li>DH10B E. coli: (F– mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara leu) 7697 galU galK rpsL nupG λ–)</li>
+ <li>JW2880-1 strain: (F-Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, ΔserA764::kanR, rph-1, Δ(rhaD-rhaB)568, hsdR514)</li>
+ </ul>
+ </li>
+ <li><strong>Saccharomyces cerevisiae:</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>S. cerevisiae Ï+ NB97: (MATa leu2-3,112 lys2 ura3-52 his3ΔHindIII arg8Δ::URA3 [cox2-60::ARG8m])</li>
+ <li>S. cerevisiae Ïo MTCC109: (ATCC201440)</li>
+ </ul>
+ </li>
+ </ul>
+ </li>
+ <li><strong>Growth Media</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li><strong>E. coli:</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>LB medium (no antibiotics)</li>
+ <li>2YT medium</li>
+ <li>Minimal agar medium (M9 medium with Casamino Acids – Vitamin Assay)</li>
+ </ul>
+ </li>
+ <li><strong>S. cerevisiae:</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>YPD medium (1% Bacto yeast extract, 2% Bacto peptone, 2% glucose)</li>
+ <li>Modified YPD (with 0.1% glucose/3% glycerol or 3% glycerol, and 1 M sorbitol)</li>
+ <li>YPDS medium (YPD medium supplemented with 1 M sorbitol)</li>
+ </ul>
+ </li>
+ </ul>
+ </li>
+ <li><strong>Selection Media</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>Selection Medium II: 1% Bacto yeast extract, 2% Bacto peptone, 3% glycerol, 0.1% glucose, 1 M sorbitol, 1.5% agar, 1 mM arabinose</li>
+ <li>Selection Medium III: 1% Bacto yeast extract, 2% Bacto peptone, 3% glycerol, 1 M sorbitol, 1.5% agar, 1 mM arabinose (without glucose)</li>
+ </ul>
+ </li>
+ <li><strong>Reagents</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>Bacterial resuspension buffer: 10 mM Tris-HCl, 2.5 mM MgCl2, 10 mM CaCl2, pH 8</li>
+ <li>PEG buffer: 20% PEG 8000, 10 mM Tris-HCl, 2.5 mM MgCl2, 10 mM CaCl2, pH 8</li>
+ <li>TSC buffer: 10 mM Tris-HCl, 10 mM CaCl2, 1 M sorbitol, pH 7.5</li>
+ <li>1 M sorbitol solution</li>
+ <li>4 M sorbitol solution</li>
+ <li>Zymolyase 100T</li>
+ </ul>
+ </li>
+ <li><strong>Additives and Antibiotics</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>10 µM thiamin</li>
+ <li>100 µM NAD</li>
+ <li>50 mg/L kanamycin</li>
+ <li>50 mg/L chloramphenicol</li>
+ <li>5 mg/L tetracycline</li>
+ <li>1 mM arabinose</li>
+ </ul>
+ </li>
+ </ul>
+
<div class="h3">Equipment</div>
<ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
- <li></li>
- </ul>
+ <li>Incubator/shaker (30°C and 37°C)</li>
+ <li>Water bath (37°C)</li>
+ <li>Centrifuge (capable of 1,500g, temperature control)</li>
+ <li>Spectrophotometer (for OD600 measurements)</li>
+ <li>Sterile culture tubes and flasks</li>
+ <li>Microcentrifuge tubes</li>
+ <li>Petri dishes</li>
+ <li>Pipettes and tips</li>
+ <li>Vortex mixer</li>
+ <li>Refrigerator (4°C)</li>
+ <li>Laminar flow hood or Bunsen burner (for sterile technique)</li>
+ </ul>
<div class="h3">Procedure</div>
<ol style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
- <li></li>
+ <li><strong>Preparation of E. coli Cells:</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>Grow E. coli cells (expressing GFP) in 5 mL of LB medium until OD600 ~ 0.8.</li>
+ <li>Harvest the cells at 4°C and wash twice with chilled bacterial resuspension buffer (10 mM Tris-HCl, 2.5 mM MgCl2, 10 mM CaCl2, pH 8).</li>
+ <li>Resuspend the cells in 500 µL of resuspension buffer.</li>
+ </ul>
+ </li>
+
+ <li><strong>Preparation of Yeast Spheroplasts:</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>Grow yeast cells in YPD medium overnight.</li>
+ <li>Harvest the cells and wash twice with sterile water, then twice with 1 M sorbitol solution (20 mL per gram of cells).</li>
+ <li>Resuspend the cells in sterile-filtered 1 M sorbitol solution (5 mL per gram of cells) containing Zymolyase 100T (5-10 mg/g of cells).</li>
+ <li>Incubate the suspension in a water bath at 37°C for 1 hour to generate spheroplasts.</li>
+ <li>Cool the spheroplast suspension on ice for 20-30 minutes, then centrifuge at 1,500g, 4°C for 10 minutes.</li>
+ <li>Wash the spheroplasts twice with 1 M sorbitol solution (5 mL per gram of cells) and resuspend in 1 M sorbitol solution (2 mL per gram of cells).</li>
+ </ul>
+ </li>
+
+ <li><strong>Fusion Procedure:</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>Mix 500 µL of spheroplast suspension with 500 µL of TSC buffer, incubate at 30°C for 10 minutes, then centrifuge at 1,500g for 10 minutes.</li>
+ <li>Resuspend the spheroplasts in 100 µL of TSC buffer.</li>
+ <li>Mix 150 µL of bacterial cell suspension quickly with 50 µL of 4 M sorbitol, then immediately add the mixture to 100 µL of spheroplast suspension in TSC buffer.</li>
+ <li>Invert the tubes to mix and incubate at 30°C for 10 minutes.</li>
+ <li>Add the mixture to 2.5 mL of PEG buffer and incubate at 30°C for 45 minutes.</li>
+ <li>Centrifuge at 1,500g, 25°C for 10 minutes. Discard the supernatant PEG solution.</li>
+ </ul>
+ </li>
+
+ <li><strong>Post-Fusion Processing:</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>Slowly add 1 mL of YPDS medium to the cell pellet without disturbing the pellet and incubate at 30°C for 2 hours.</li>
+ <li>Partially resuspend the pellet by tapping the side of the tube and incubate in a 30°C shaker at 70 rpm for 3 hours.</li>
+ <li>Pellet the cells by centrifugation at 1,500g at room temperature, resuspend in 1 M sorbitol, and plate on selection medium II.</li>
+ <li>Overlay the plated cells with the same selection medium preincubated at 50°C (1.5% agar).</li>
+ <li>Incubate the plates at 25°C for 3-4 days.</li>
+ </ul>
+ </li>
+
+ <li><strong>Subsequent Culturing:</strong>
+ <ul style="text-align:justify; font-family:AccidenzCommons; color:#185A4F; font-weight:400; font-size: min(1.5vw, 22px); font-style: normal; line-height: normal;">
+ <li>For subsequent rounds of growth, plate the cells on selection medium II or selection medium III as indicated.</li>
+ </ul>
+ </li>
</ol>
+
</div>
+
<!-- 9 -->
<div class="h" id="nine">
<div class="h1">References</div>
--
GitLab