Difference between revisions of "Team:Heidelberg/Notebook"

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{{Heidelberg/header}}
 
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{{#tag:html|<script>{{Heidelberg/title|Methods}}</script>}}
 
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{{Heidelberg/main|Notebook|Methods|https://static.igem.org/mediawiki/2017/5/50/T--Heidelberg--2017_notebook-bg.png|orange|
 
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{{Heidelberg/templateus/Contentsection|{{Heidelberg/accpanel|#F8991D|{{Heidelberg/accord|Agarose-Gelelectrophoresis|{{Heidelberg/templateus/Tablebox||{{#tag:html|<h2 id="-agarose-gelelectrophoresis">Agarose-Gelelectrophoresis</h1>
 
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<ol>
 
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<li>Prepare a 1 % agarose-gel: Dissolve the agarose in 1X TAE by boiling and add ethidium bromide (5 µl per 100 ml agarose solution).<br>For short DNA fragments, gels with higher agarose percentage should be used.
 
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</li>
 
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<li>Pour the gel and let it polymerize until it is solid.
 
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</li>
 
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<li>Add the appropriate amount of 6X purple loading dye (NEB) to the PCR or digestion reaction mix (8 µl for 50 µl PCR reaction).
 
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</li>
 
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<li>Place the gel into the tank and add 1X TAE buffer so that the gel is fully covered.
 
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</li>
 
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<li>Pipet the DNA into the pockets.
 
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</li>
 
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<li>Run the gel at 120 V for 20 min.
 
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</li>
 
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<li>Image the gel using ultraviolet light. If necessary, cut out bands.
 
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</li>
 
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</ol>
 
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}}|
 
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}}|table_id-0}}
 
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{{Heidelberg/accord|Beta-Galactosidase Activity Assay|{{Heidelberg/templateus/Tablebox||{{#tag:html|<h2 id="-galactosidase-activity-assay">ß-Galactosidase Activity Assay</h1>
 
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To determine the activities of the wildtype ß-galactosidase and the ß-glucuronidase mutants, an assay with p-nitrophenyl-galactopyranoside (PNPG) was setup.
 
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1X assay buffer consisted of the following substances:
 
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{{Heidelberg/templateus/Tablebox||{{#tag:html|<table class="table table-bordered mdl-shadow--4dp" XSSCleaned="overflow-x: scroll !important">
 
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<thead>
 
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<tr>
 
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<th>Chemical</th>
 
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<th>Conentration [mM]</th>
 
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</tr>
 
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</thead>
 
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<tbody>
 
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<tr>
 
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<td>Disodium phosphate</td>
 
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<td>100</td>
 
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</tr>
 
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<tr>
 
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<td>Magnesium chloride</td>
 
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<td>1</td>
 
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</tr>
 
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</tbody>
 
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</table>}}|}}
 
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Beta-mercaptoethanol|50|
 
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PNPG was diluted in assay buffer in seven different concentrations: 3 mM, 1.5 mM, 0.75 mM, 0.375 mM, 0.188 mM, 0.094 mM, 0.047 mM. 10 µl of the different substrate solutions were prepared in a 96 well microtitre plate. In the last well of each column, 10 µl assay buffer were used as blank.
 
−
The enzymes were diluted in assay buffer to a final concentration of 4.31 mM. 90 µl of enzyme solution were added to the substrate with a multichannel pipette. The reaction was followed in a plate reader for 45 min. Every minute, absorbance was measured at 420 nm. The experiment was carried out in triplicates. From the results, the Michaelis Menten values Km, Vmax and Kcat could be determined.
 
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}}|
 
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}}|table_id-1}}
 
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{{Heidelberg/accord|Beta-Galactosidase assay on Agar-plates|{{Heidelberg/templateus/Tablebox||{{#tag:html|ß-Galectosidase assay on plates
 
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To generate a visual output for ß-Galactosidase activity of the ß-glucuronidase (GUS) mutants, a 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) assay was set up. Therefore, LB-agar plates with 100 µg/ml kanamycin, 4 mg/ml X-Gal and 0.5 mM IPTG were prepared. Cultures with the different GUS mutants and the wiltype enzyme were grown overnight to the stationary phase. The next day, 10 µl of each culture were plated in eighths on the same plates. When the blue color started to develop, the plates were imaged.
 
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}}|
 
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}}|table_id-2}}
 
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{{Heidelberg/accord|Blue Plaque Assay|{{Heidelberg/templateus/Tablebox||{{#tag:html|<h2 id="blue-plaque-assay-170801-">Blue Plaque Assay (170801)</h1>
 
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<h2 id="1-introduction">1. Introduction</h2>
 
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The Blue Plaque Assay is a method to determine M13 phage titer.
 
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<h2 id="2-material">2. Material</h2>
 
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<strong>LB/Amp-Agar Plates (1.5 % agar):</strong><br>Plates with bottom agar (15 g/L agar in LB medium) supplemented with antibiotic are prepared at least 30 min before plaque assay.
 
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<strong>Top Agar (0.7 % agar):</strong><br>Top agar containing 7 g/l agar is autoclaved and stored at ~56 °C in the heating cabinet. 3 ml top agar is sufficient for one agar plate.
 
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<strong>X-gal stock solution (40 mg/ml):</strong><br> 0.2 g X-gal is dissolved in 5 ml DMSO and stored at -20 °C in the dark. It is useful to aliquot the stock solution in 1.5-ml reaction tubes.
 
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<strong>Transformed S2060 <em>E. coli</em> strain (ID:47):</strong><br>S2060 <em>E. coli</em> possess the lacZ gene under control of a PSP promoter. For the Blue Plaque Assay a transformed S2060 strain like <em>E. coli</em> strain ID:47 containing the AP of the S1059 strain (gene III under a PSP promoter) are used.
 
−
(This protocol can be modified to investigate if the evolving phage can resist stronger selection pressure. Therefore S2060 strain has to be transformed with the associated AP and the antibiotics has to be adapted)
 
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<h2 id="3-procedure">3. Procedure</h2>
 
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<strong>Day 1:</strong>
 
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S2060 AP_PSP_gIII cells (ID:47) or the appropriate cell type are used to inoculate 4 mL of 2xYT medium containing 25 mM glucose and carbenicillin or the necessary antibiotic for that cell type.<br>The culture is placed in the 37°C shaker in the morning and are grown until an  OD600 of 0.6-0.8.
 
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In the meantime, 10 to 10<sup>2</sup>-fold serial dilutions of the phage suspension are prepared with LB medium. 1 ml final volumes are convenient. To prevent cross-contamination filter tips must be used. The phage samples can be stored at 4&nbsp;°C for a long time and still be able to infect <em>E. coli</em> cells.
 
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Shortly before the <em>E. coli</em> culture reaches the appropriate OD600 top agar (3&nbsp;ml per plate) is transferred to a 50-ml falcon and placed in a preheated water bath at 50&nbsp;°C. Top agar is then supplemented with the necessary antibiotic and the x-gal stock solution (40&nbsp;mg/ml).
 
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For 10 ml top agar:
 
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<ul>
 
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<li>10 µl antibiotic (1000x)</li>
 
−
<li>100 µl X-gal stock solution (100x)</li>
 
−
</ul>
 
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As soon as the <em>E. coli</em> culture reach an OD600 of 0.6-0.8, cell suspension is gently mixed by resuspension with a pipette and 50&nbsp;µl are transferred to a 1.5-ml reaction tube for each condition. The following steps has to be performed quickly and separate for each condition. Phage suspension is shortly vortexed and 1&nbsp;µl is pipetted to the <em>E. coli</em> culture. 700 µl of the top agar is thorough mixed with the cell and phage suspension. This is immediately poured on the corresponding quarter of the plate and rocked gently to evenly spread it.
 
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After allowing the top agar to solidify for few minutes, plates were sealed with parafilm and incubated overnight at 37°C.
 
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<strong>Day 2:</strong>
 
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On the next day blue plaques can be counted. For quantification of the phage titer the number of plaques should be between 10 and 100.<br>The amount of phages in the solution is calculated by:
 
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PFU/ml = number of plaques x dilution factor x 1000
 
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e.g. 12 blue plaques are counted on a plate quarter where 1&nbsp;µl of a 10<sup>-4</sup> phage dilution was plated.<br>12 x 10<sup>4</sup> x 1000 = 1.2 x 10<sup>8</sup> PFU/ml
 
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}}|
 
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}}|table_id-3}}
 
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{{Heidelberg/accord|Chemical Competent Cells|{{Heidelberg/templateus/Tablebox||{{#tag:html|Preparation of Calcium Chloride Competent <em>E. coli</em> Cells====1. Inoculate 5 ml LB with a colony of the respective strain and let it grow overnight at 37 °C at 220 rpm. If the strain carries a plasmid, do not forget to add the respective antibiotics. 2. The next day, inoculate 250 ml LB medium with 1 ml of the overnight culture and let it grow until it reaches an OD600 of 0.6-0.7. * Work on ice from now on. 3. Cool the cells on ice for 10 min. 4. Centrifuge the cells for 10 min at 6000 g and discard the supernatant. 5. Resuspend the pellet in 10 ml of pre cooled 0.1 M CaCl. 6. Incubate the suspension for 20 min on ice. 7. Centrifuge again for 10 min at 6000 g and discard the supernatant. 8. Resuspend the pellet in 5 ml of cold 0.1 M CaCl with 15% Glycerol 9. Freeze 50 µl aliquots of bacteria suspension in liquid nitrogen and store at -80 °C.
 
−
}}|
 
−
}}|table_id-4}}
 
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{{Heidelberg/accord|Chemical Transformation|{{Heidelberg/templateus/Tablebox||{{#tag:html|Transformation into chemical competent Cells===1. Thaw an aliquot of CaCl competent cells on ice. 2. Add 5 µl of the ligation reaction and incubate for 30 min on ice. 3. Heat shock the cells for 1 min at 42 °C. 4. Incubate on ice for 2 min. 5. Add 1 ml of SOC and incubate for 1 h at 37 °C on a shaker. 6. Plate the appropriate amount of cells on agar with the respective antibiotic.
 
−
}}|
 
−
}}|table_id-5}}
 
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{{Heidelberg/accord|Circular Polymerase Extension Cloning|{{Heidelberg/templateus/Tablebox||{{#tag:html|Circular Polymerase Extension Cloning (CPEC)
 
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<ol>
 
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<li>Mix 75 ng of backbone with equimolar amounts of inserts (short fragments can be used in excess) and add 25 µl Phusion Flash Master Mix (Thermo Fisher Scientific). Fill up to 50 µl with dH2O.
 
−
</li>
 
−
<li>Run the polymerase chain reaction in a thermocycler with the following protocol:
 
−
</li>
 
−
</ol>
 
−
{{Heidelberg/templateus/Tablebox||{{#tag:html|<table class="table table-bordered mdl-shadow--4dp" XSSCleaned="overflow-x: scroll !important">
 
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<thead>
 
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<tr>
 
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<th>Step</th>
 
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<th>Temperature</th>
 
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<th>Time</th>
 
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</tr>
 
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</thead>
 
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<tbody>
 
−
<tr>
 
−
<td>1</td>
 
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<td>98 °C</td>
 
−
<td>10 s</td>
 
−
</tr>
 
−
<tr>
 
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<td>2</td>
 
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<td>98 °C</td>
 
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<td>1 s</td>
 
−
</tr>
 
−
<tr>
 
−
<td></td>
 
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<td>slow ramp</td>
 
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<td>0.1 °C/s</td>
 
−
</tr>
 
−
<tr>
 
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<td>3</td>
 
−
<td>55 °C</td>
 
−
<td>5 s</td>
 
−
</tr>
 
−
<tr>
 
−
<td>4</td>
 
−
<td>72 °C</td>
 
−
<td>15-30 s/kb</td>
 
−
</tr>
 
−
<tr>
 
−
<td>5</td>
 
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<td>72 °C</td>
 
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<td>5 min</td>
 
−
</tr>
 
−
<tr>
 
−
<td>repeat step 2-4 25x</td>
 
−
</tr>
 
−
</tbody>
 
−
</table>}}|}}
 
−
<ol>
 
−
<li>Transform a few µl of the reaction into a suitable bacteria strain</li>
 
−
</ol>
 
−
}}|
 
−
}}|table_id-6}}
 
−
{{Heidelberg/accord|DNA Preparation|{{Heidelberg/templateus/Tablebox||{{#tag:html|<h2 id="miniprep-pcr-purification-gel-extraction">Miniprep, PCR Purification, Gel Extraction</h1>
 
−
For DNA preparation, kits provided by Qiagen were used according to the manufacturer&#39;s instructions. DNA concentration was determined using a nanophotometer.
 
−
}}|
 
−
}}|table_id-7}}
 
−
{{Heidelberg/accord|Electrocompetent Cells|{{Heidelberg/templateus/Tablebox||{{#tag:html|Preparation of electrocompetent <em>E. coli</em> cells
 
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<h2 id="preparation-">Preparation:</h2>
 
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Autoclave the following solutions:
 
−
Low salt media:    4.5 g Tryptone, 2.25 g Yeast Extract, 1.8 g NaCl in 450 ml dH2O<br>dH2O:              1 l<br>10 % Glycerol:      400 ml
 
−
Procedure:
 
−
<ol>
 
−
<li>Prepare an overnight culture of the desired <em>E. coli</em> strain
 
−
</li>
 
−
<li>The next morning, inoculate the low salt medium with the overnight culture
 
−
</li>
 
−
<li>Let the culture grow until an OD600 of 0.6-0.7
 
−
</li>
 
−
</ol>
 
−
5 is reached. Chill the flask on ice for 15 min
 
−
<ol>
 
−
<li>Centrifuge the bacteria for 20 min at 3000 g at 4 °C
 
−
</li>
 
−
<li>Discard the supernatant and resuspend it in 400 ml cold H2O
 
−
</li>
 
−
<li>Repeat the washing step9. Centrifuge the bacteria for 20 min at 3000 g at 4 °C and discard the remaining water
 
−
</li>
 
−
<li>Resuspend the cells in 400 ml glycerol
 
−
</li>
 
−
<li>Centrifuge agein at 3000 g for 20 min (4 °C)
 
−
</li>
 
−
<li>Discard the supernatant and resuspend the pellet in approximately 1 ml of remaining glycerol.
 
−
</li>
 
−
<li>Freeze 50 µl aliquots in liquid nitrogen and store them at -80 °C
 
−
</li>
 
−
</ol>
 
−
}}|
 
−
}}|table_id-8}}
 
−
{{Heidelberg/accord|Electroporation|{{Heidelberg/templateus/Tablebox||{{#tag:html|Transformation of <em>E. coli</em> via electroporation
 
−
Preparation:
 
−
<ol>
 
−
<li>Pipet 1 µl of the plasmid solution into a pre-cooled 1.5 ml reaction tube. Ligation products have to be diluted in H2O (1:4 usually works) or purified in advance.
 
−
</li>
 
−
<li>Heat 1 ml of SOC to 37 °C.
 
−
</li>
 
−
<li>Pre-cool an 1 mm electroporation cuvette on ice.
 
−
</li>
 
−
<li>Thaw an aliquot of electrocompetent cells on ice.
 
−
</li>
 
−
</ol>
 
−
<h2 id="procedure-">Procedure:</h2>
 
−
<ol>
 
−
<li>Pipet 25 µl of the competent cells into the tube with the plasmid solution.
 
−
</li>
 
−
<li>Transfer the mixture carefully into the cuvette (avoid air bubbles)
 
−
</li>
 
−
</ol>
 
−
.3. Electroporate at 1800 kV for 5 ms.
 
−
<ol>
 
−
<li>Immediately add 1 ml SOC, mix by pipetting up and down, and transfer the solution into a 1.5 ml reaction tube
 
−
</li>
 
−
<li>Incubate for 1 h at 37 °C at 350 rpm
 
−
</li>
 
−
</ol>
 
−
.6. Plate 100 µl on agar with the respective antibiotics.
 
−
Note: If more than one plasmid has to be transformed, the procedure stays the same. Both plasmids have to be mixed in equal amount prior to the addition to the cells. Again, only 1 µl should be used. For the last step, agar plates with 2 antibiotics have to be used.
 
−
}}|
 
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}}|table_id-9}}
 
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{{Heidelberg/accord|Gibson Assembly|{{Heidelberg/templateus/Tablebox||{{#tag:html|<h2 id="-gibson-assembly">Gibson Assembly</h1>
 
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Preparation of Gibson Assembly mastermix using the protocol provided by the Miller lab:
 
−
Preparation of 5X ISO-buffer:
 
−
{{Heidelberg/templateus/Tablebox||{{#tag:html|<table class="table table-bordered mdl-shadow--4dp" XSSCleaned="overflow-x: scroll !important">
 
−
<thead>
 
−
<tr>
 
−
<th style="text-align:right">Component</th>
 
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<th style="text-align:right">Amount</th>
 
−
</tr>
 
−
</thead>
 
−
<tbody>
 
−
<tr>
 
−
<td style="text-align:right">1 M Tris-HCl pH 7.5</td>
 
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<td style="text-align:right">3 ml</td>
 
−
</tr>
 
−
<tr>
 
−
<td style="text-align:right">2 M MgCl2</td>
 
−
<td style="text-align:right">150 µl</td>
 
−
</tr>
 
−
<tr>
 
−
<td style="text-align:right">100 nM dNTP mix</td>
 
−
<td style="text-align:right">60 µl</td>
 
−
</tr>
 
−
<tr>
 
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<td style="text-align:right">1 M DTT</td>
 
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<td style="text-align:right">300µl</td>
 
−
</tr>
 
−
<tr>
 
−
<td style="text-align:right">PEG-8000</td>
 
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<td style="text-align:right">1.5 g</td>
 
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</tr>
 
−
<tr>
 
−
<td style="text-align:right">100 mM NAD</td>
 
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<td style="text-align:right">300 µl</td>
 
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</tr>
 
−
<tr>
 
−
<td style="text-align:right">dH2O</td>
 
−
<td style="text-align:right">add to 6 ml</td>
 
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</tr>
 
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</tbody>
 
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</table>}}|}}
 
−
Preparation of Gibson Assembly mastermix:
 
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{{Heidelberg/templateus/Tablebox||{{#tag:html|<table class="table table-bordered mdl-shadow--4dp" XSSCleaned="overflow-x: scroll !important">
 
−
<thead>
 
−
<tr>
 
−
<th style="text-align:right">Component</th>
 
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<th style="text-align:right">Amount</th>
 
−
</tr>
 
−
</thead>
 
−
<tbody>
 
−
<tr>
 
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<td style="text-align:right">5X ISO buffer</td>
 
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<td style="text-align:right">320 µl</td>
 
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</tr>
 
−
<tr>
 
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<td style="text-align:right">10U/µl T5 exonuclease</td>
 
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<td style="text-align:right">0.64 µl</td>
 
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</tr>
 
−
<tr>
 
−
<td style="text-align:right">2 U/µl Phusion ploymerase</td>
 
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<td style="text-align:right">20 µl</td>
 
−
</tr>
 
−
<tr>
 
−
<td style="text-align:right">40 U/µl Taq ligase</td>
 
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<td style="text-align:right">160 µl</td>
 
−
</tr>
 
−
<tr>
 
−
<td style="text-align:right">dH2O</td>
 
−
<td style="text-align:right">add to 1.2 ml</td>
 
−
</tr>
 
−
</tbody>
 
−
</table>}}|}}
 
−
<h2 id="procedure-">Procedure:</h2>
 
−
<ol>
 
−
<li>Mix 10 µl Gibson Assembly mastermix with 0.02–0.5 pmol of backbone and equimolar amounts of insert.
 
−
</li>
 
−
<li>Incubate at 50 °C for 15-60 min
 
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</li>
 
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</ol>
 
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.3. Use 5 µl for transformation in CaCl competent cells.
 
−
}}|
 
−
}}|table_id-10}}
 
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{{Heidelberg/accord|Glycerol-Stocks|{{Heidelberg/templateus/Tablebox||{{#tag:html|<h2 id="-glycerol-stocks">Glycerol Stocks</h1>
 
−
<ol>
 
−
<li>Prepare a 50 % glycerol solution diluted in H2O and autoclave it.
 
−
</li>
 
−
<li>Mix 500 µl of an overnight culture with 500 µl of 50 % glycerol solution, and store it at -80 °C.
 
−
</li>
 
−
</ol>
 
−
}}|
 
−
}}|table_id-11}}
 
−
{{Heidelberg/accord|Golden Gate|{{Heidelberg/templateus/Tablebox||{{#tag:html|<h2 id="-golden-gate-assembly">Golden Gate Assembly</h1>
 
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Prepare the following reaction mix with a total volume of 15 µl:
 
−
{{Heidelberg/templateus/Tablebox||{{#tag:html|<table class="table table-bordered mdl-shadow--4dp" XSSCleaned="overflow-x: scroll !important">
 
−
<thead>
 
−
<tr>
 
−
<th>Component</th>
 
−
<th>Volume [µl]</th>
 
−
</tr>
 
−
</thead>
 
−
<tbody>
 
−
<tr>
 
−
<td>T4 DNA Ligase (NEB)</td>
 
−
<td>1</td>
 
−
</tr>
 
−
<tr>
 
−
<td>10x T4 DNA Ligase Reaction Buffer (NEB)</td>
 
−
<td>1.5</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Type IIS cutter</td>
 
−
<td>1</td>
 
−
</tr>
 
−
<tr>
 
−
<td>DNA fragment</td>
 
−
<td>X</td>
 
−
</tr>
 
−
<tr>
 
−
<td>dH2O</td>
 
−
<td>adjust to 15 µl</td>
 
−
</tr>
 
−
</tbody>
 
−
</table>}}|}}
 
−
For our project, we used the restriction enzymes BsaI-HF (NEB), BbsI-HF (NEB), and Esp3I (Thermo Fisher Scientific) for Golden Gate Assembly.
 
−
75 ng of backbone DNA is mixed with and equimolar amount of inserts. If an insert is shorter than 500 bp, it was added in 2:1 excess.For the polymerase chain reaction the following programm was used in a thermocycler:
 
−
{{Heidelberg/templateus/Tablebox||{{#tag:html|<table class="table table-bordered mdl-shadow--4dp" XSSCleaned="overflow-x: scroll !important">
 
−
<thead>
 
−
<tr>
 
−
<th>Step</th>
 
−
<th>Temperature</th>
 
−
<th>Time</th>
 
−
</tr>
 
−
</thead>
 
−
<tbody>
 
−
<tr>
 
−
<td>1</td>
 
−
<td>37 °C</td>
 
−
<td>2 min</td>
 
−
</tr>
 
−
<tr>
 
−
<td>2</td>
 
−
<td>16 °C</td>
 
−
<td>3 min</td>
 
−
</tr>
 
−
<tr>
 
−
<td>3</td>
 
−
<td>16 °C</td>
 
−
<td>5 min</td>
 
−
</tr>
 
−
<tr>
 
−
<td>4</td>
 
−
<td>55 °C</td>
 
−
<td>5 min</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Repeat step one and two 20x</td>
 
−
</tr>
 
−
</tbody>
 
−
</table>}}|}}
 
−
.Use 5 µl of the reaction mix for a chemical transformation<br>.
 
−
}}|
 
−
}}|table_id-12}}
 
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{{Heidelberg/accord|Ligations|{{Heidelberg/templateus/Tablebox||{{#tag:html|<h2 id="-ligation">Ligation</h1>
 
−
<ol>
 
−
<li>Assemble the reaction mix as listed in the table below:</li>
 
−
</ol>
 
−
{{Heidelberg/templateus/Tablebox||{{#tag:html|<table class="table table-bordered mdl-shadow--4dp" XSSCleaned="overflow-x: scroll !important">
 
−
<thead>
 
−
<tr>
 
−
<th>Component</th>
 
−
<th>Volume</th>
 
−
</tr>
 
−
</thead>
 
−
<tbody>
 
−
<tr>
 
−
<td>T4 DNA Ligase (NEB)</td>
 
−
<td>1 µl</td>
 
−
</tr>
 
−
<tr>
 
−
<td>10x T4 DNA Ligase Reaction Buffer (NEB)</td>
 
−
<td>1.5 µl</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Vector backbone</td>
 
−
<td>50 ng</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Insert</td>
 
−
<td>add in 3:1 molar ratio</td>
 
−
</tr>
 
−
<tr>
 
−
<td>dH2O</td>
 
−
<td>adjust to 15 µl</td>
 
−
</tr>
 
−
</tbody>
 
−
</table>}}|}}
 
−
<ol>
 
−
<li>Incubate at room temperature for 10 min or overnight at 16 °C.
 
−
</li>
 
−
<li>Heat inactivate the ligase at 65 °C for 10 min
 
−
</li>
 
−
</ol>
 
−
.4. Use a few µl for transformation into competent cells.
 
−
}}|
 
−
}}|table_id-13}}
 
−
{{Heidelberg/accord|Overnight-Culture|{{Heidelberg/templateus/Tablebox||{{#tag:html|<h2 id="-overnight-cultures-">Overnight cultures:</h1>
 
−
<ol>
 
−
<li>Prepare LB media by resolving 25 g LB-powder (Roth) in 1 l of dH2O, and autoclave it.
 
−
</li>
 
−
<li>Transfer 4 ml of LB into a culture tube, and add 4 µl of the respective antibiotic stock solutions
 
−
</li>
 
−
</ol>
 
−
.3. Inoculate the culture by picking a single colony with a pipet tip and tipping it into the medium or by adding a few microliters from a glycerol-stock.
 
−
<ol>
 
−
<li>Shake the culture at 37 °C at 220 rpm for 12-14 h.
 
−
</li>
 
−
<li>Isolate plasmids or use the culture for further experiments.
 
−
</li>
 
−
</ol>
 
−
Concentrations of antibiotics:
 
−
{{Heidelberg/templateus/Tablebox||{{#tag:html|<table class="table table-bordered mdl-shadow--4dp" XSSCleaned="overflow-x: scroll !important">
 
−
<thead>
 
−
<tr>
 
−
<th>Antibiotic</th>
 
−
<th>Stock Concentration (1000X)</th>
 
−
<th>Final Concentration</th>
 
−
<th></th>
 
−
</tr>
 
−
</thead>
 
−
<tbody>
 
−
<tr>
 
−
<td>Ampicillin</td>
 
−
<td>100 mg/ml</td>
 
−
<td>100 µg/ml</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Streptomycin</td>
 
−
<td>100 mg/ml</td>
 
−
<td>100 µg/ml</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Kanamycin</td>
 
−
<td>50 mg/ml</td>
 
−
<td>50 µg/ml</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Chloramphenicol</td>
 
−
<td>25 mg/ml</td>
 
−
<td>25 µg/ml</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Spectinomycin</td>
 
−
<td>50 mg/ml</td>
 
−
<td>50 µg/ml</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Carbenicillin</td>
 
−
<td>100 mg/ml</td>
 
−
<td>100 µg/ml</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Tetracyclin</td>
 
−
<td>10 mg/ml</td>
 
−
<td>10 µg/ml</td>
 
−
</tr>
 
−
</tbody>
 
−
</table>}}|}}
 
−
}}|
 
−
}}|table_id-14}}
 
−
{{Heidelberg/accord|Plates|{{Heidelberg/templateus/Tablebox||{{#tag:html|<h2 id="-agar-plates">Agar plates</h1>
 
−
<ol>
 
−
<li>For 1 l of 1.5 % agar resolve 15 g Agar-Agar (Roth) and 25 g LB-powder (Roth) in 1 l dH2O, and autoclave it.
 
−
</li>
 
−
<li>Wait until the agar cools down to ~ 50 °C, and then add the respective antibiotic (see table below).
 
−
</li>
 
−
<li>Pour 8.5 cm plates with 15-18 ml of agar.
 
−
</li>
 
−
<li>Store the plates at 4 °C.
 
−
</li>
 
−
<li>Streak the bacteria out, and incubate at 37 °C for 12-16 h.
 
−
</li>
 
−
</ol>
 
−
Antibiotic-concentrations:
 
−
{{Heidelberg/templateus/Tablebox||{{#tag:html|<table class="table table-bordered mdl-shadow--4dp" XSSCleaned="overflow-x: scroll !important">
 
−
<thead>
 
−
<tr>
 
−
<th>Antibiotic</th>
 
−
<th>Stock Concentration</th>
 
−
<th>Final Concentration</th>
 
−
<th></th>
 
−
</tr>
 
−
</thead>
 
−
<tbody>
 
−
<tr>
 
−
<td>Ampicillin</td>
 
−
<td>100 mg/ml</td>
 
−
<td>100 µg/ml</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Streptomycin</td>
 
−
<td>100 mg/ml</td>
 
−
<td>100 µg/ml</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Kanamycin</td>
 
−
<td>50 mg/ml</td>
 
−
<td>50 µg/ml</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Chloramphenicol</td>
 
−
<td>25 mg/ml</td>
 
−
<td>25 µg/ml</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Spectinomycin</td>
 
−
<td>50 mg/ml</td>
 
−
<td>50 µg/ml</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Carbenicillin</td>
 
−
<td>100 mg/ml</td>
 
−
<td>100 µg/ml</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Tetracyclin</td>
 
−
<td>10 mg/ml</td>
 
−
<td>10 µg/ml</td>
 
−
</tr>
 
−
</tbody>
 
−
</table>}}|}}
 
−
}}|
 
−
}}|table_id-15}}
 
−
{{Heidelberg/accord|Polymerase Chain Assembly|{{Heidelberg/templateus/Tablebox||{{#tag:html|<h2 id="-polymerase-chain-assembly">Polymerase Chain Assembly</h1>
 
−
<ol>
 
−
<li>Mix the DNA fragments in equimolar ratio. The total amount of DNA should be between 10 ng and 15 ng. Add 10 µl of 2X Phusion Flash Master Mix (Thermo Fisher Scientific), and adjust to 20 µl with dH2O.
 
−
</li>
 
−
<li>Run the following program:
 
−
</li>
 
−
</ol>
 
−
{{Heidelberg/templateus/Tablebox||{{#tag:html|<table class="table table-bordered mdl-shadow--4dp" XSSCleaned="overflow-x: scroll !important">
 
−
<thead>
 
−
<tr>
 
−
<th>Step</th>
 
−
<th>Temperature</th>
 
−
<th>Time</th>
 
−
</tr>
 
−
</thead>
 
−
<tbody>
 
−
<tr>
 
−
<td>1</td>
 
−
<td>98 °C</td>
 
−
<td>10 s</td>
 
−
</tr>
 
−
<tr>
 
−
<td>2</td>
 
−
<td>98 °C</td>
 
−
<td>1 s</td>
 
−
</tr>
 
−
<tr>
 
−
<td>3</td>
 
−
<td>55 °C</td>
 
−
<td>5 s</td>
 
−
</tr>
 
−
<tr>
 
−
<td>4</td>
 
−
<td>72 °C</td>
 
−
<td>15-30 s/kb</td>
 
−
</tr>
 
−
<tr>
 
−
<td>5</td>
 
−
<td>72 °C</td>
 
−
<td>5 min</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Repeat step 2-4 10x</td>
 
−
</tr>
 
−
</tbody>
 
−
</table>}}|}}
 
−
<ol>
 
−
<li>Add 5 µl of the reaction to 25 µl Phusion Flash Master mix. Add primers to a final concentration of 0.5 µM, and add dH2O to a final volume of 50 µl.
 
−
</li>
 
−
<li>Cycle again using the following conditions:
 
−
</li>
 
−
</ol>
 
−
{{Heidelberg/templateus/Tablebox||{{#tag:html|<table class="table table-bordered mdl-shadow--4dp" XSSCleaned="overflow-x: scroll !important">
 
−
<thead>
 
−
<tr>
 
−
<th>Step</th>
 
−
<th>Temperature</th>
 
−
<th>Time</th>
 
−
</tr>
 
−
</thead>
 
−
<tbody>
 
−
<tr>
 
−
<td>1</td>
 
−
<td>98 °C</td>
 
−
<td>10 s</td>
 
−
</tr>
 
−
<tr>
 
−
<td>2</td>
 
−
<td>98 °C</td>
 
−
<td>1 s</td>
 
−
</tr>
 
−
<tr>
 
−
<td>3</td>
 
−
<td>Annealing Tm °C</td>
 
−
<td>5 s</td>
 
−
</tr>
 
−
<tr>
 
−
<td>4</td>
 
−
<td>72 °C</td>
 
−
<td>15-30 s/kb</td>
 
−
</tr>
 
−
<tr>
 
−
<td>5</td>
 
−
<td>72 °C</td>
 
−
<td>5 min</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Repeat step 2-4 20-30x</td>
 
−
</tr>
 
−
</tbody>
 
−
</table>}}|}}
 
−
}}|
 
−
}}|table_id-16}}
 
−
{{Heidelberg/accord|PREDCEL Protocol|{{Heidelberg/templateus/Tablebox||{{#tag:html|<h2 id="predcel-protocol">PREDCEL Protocol</h1>
 
−
<h2 id="1-introduction">1. Introduction</h2>
 
−
Phage Related DisContinuous EvoLution (PREDCEL) is a batch method to perform directed evolution adapted from PACE standing for Phage Assisted Continuous Evolution.
 
−
<h2 id="2-material">2. Material</h2>
 
−
<ul>
 
−
<li>Respective bacterial strains, usually <em>E. coli</em>, with Mutation Plasmids (MP), Accessory Plasmids (AP), and if necessary further plasmids, like Complementary Plasmid (CP)</li>
 
−
<li>Phages carrying the Selection Plasmid (SP)</li>
 
−
<li>2YT medium</li>
 
−
<li>Glucose stock solution (final concentration: 100 mM)</li>
 
−
<li>Arabinose stock solution (final concentration: 100 mM)</li>
 
−
<li>Phage buffer solution (10 ml 1 M Tris; pH 7.5; 10 ml 1 M MgSO4;<br>4 g NaCl; 980 ml ddH2O)</li>
 
−
<li>Flasks, all common sizes</li>
 
−
<li>Centrifugation tubes (2 ml and 50 ml)</li>
 
−
<li>Table top centrifuge (minimum 6000 g are required)</li>
 
−
<li>Centrifuge (minimum 3750 g are required)</li>
 
−
<li>Incubator</li>
 
−
<li>Freezer, -80 °C</li>
 
−
<li>Fridge, +4 °C</li>
 
−
</ul>
 
−
<h2 id="3-procedure">3. Procedure</h2>
 
−
As it is important to prevent phage contaminations at any time, all of the following PREDCEL steps has to be performed with filter tips, and benches must be cleaned with 10% H202 (pay attention with handling!) or another suitable solution for phage inactivation. If practicable, use UV light on benches and in incubators to sterilize after and before your experiments.
 
−
<strong>Step 1: Prove MP and AP to work</strong><br>After successfully transforming your AP and MP plasmids into your bacterial strain (see transformation protocols), but before starting your PREDCEL run, make sure whether your AP and MP work is essential for the success of the intended directed evolution process or not. If yes, follow the instructions of <em>AP testing</em> and <em>MP testing</em>.
 
−
<strong>Step 2: F+ strains</strong><br>Make sure the used strain carries the F-Pilus plasmid as it is required for proper M13 phage infection. Thus, use a bacterial culture showing a positive result in <em>Step 1</em>, and plate it on a tetracycline (tet) plate, as your strain should carry a tetracycline resistance on its F-Pilus.
 
−
<strong>Step 3: Growing culture</strong><br>Pick a clone from <em>Step 2</em>, and prepare a bacterial culture in a suitable flask filled with 20 ml or more of 2YT medium with 100 mM glucose. Glucose must be added as it prevents MP activation, and thereby keeps cells intact. Incubate the culture until an OD600 of 0.6-0.8 is reached. At this time bacterial cells are in exponential stage and can be safely infected by phages.
 
−
<strong>Step 4: Glycerol stocks</strong><br>Take 5 ml from <em>step 3</em> to prepare glycerol stocks (see respective protocol; a main stock of 1 ml and several aliquots of 100 µl), and store them at -80 °C.
 
−
<strong>Step 5: Centrifugation and MP activation</strong><br>Centrifuge 10 ml or more of culture for 10 min at 3750 g to pellet your bacterial cells. The cultures result either from <em>step 3</em> or while PREDCEL performance from glycerol stock aliquot culture with an OD600 of 0.6-0.8, that needs to be prepared early enough to minimize time between gain of phage supernatant and new infection round. If culture reaches the respective OD600 too fast, dilute the culture until you can infect with phages or cool on ice if it takes less than 10 minutes until infection. Afterwards, resolve your cell pellet in a 150 ml flask with a volume of 2YT equal to the volume spun down containing 100 mM arabinose to induce MP.
 
−
<strong>Step 6: Phage infection</strong><br>Infect with a suitable amount of phages. As titers of inoculation phage can vary a lot a MOI of 1 is recommended for PREDCEL rather than giving a specific inoculation volume. MOI of 1 means that there is one phage per cell in solution. In contrast, while performing PREDCEL, define a certain phage supernatant volume you kept at 4°C to be transferred, for instance 1 ml, as phage titer cannot be determined fast enough by plaque assays to calculate the right MOI. Make sure you have at least 1 ml of phage supernatant left to analyze. Transfer the same amount of phages used for infection into a phage buffer solution, especially if titer of inoculation phage is unknown. Further, keep 1ml of each culture before infection. They can be used later to calculate propagation efficiency and check contamination.<br>Grow the cultures for 1 to 24 hours at optimal growth conditions (for <em>E. coli</em> choose 37°C and about 220 rpm).
 
−
<strong>Step 7: Phage supernatant</strong><br>After incubation, spin down at least 2 ml of cells at 6000 g for 3 min to pellet your cells. Phages in the supernatant are now separated from the bacteria in the pellet. Store phage supernatant at 4°C. Discard the rest of the culture.
 
−
From now on repeat steps 5 to 7. After one round is completed, you can pause the PREDCEL process and restart later on with the stored supernatant.
 
−
While PREDCELing, detect and prevent phage washout or phage contamination in your used stocks, media and bacteria cultures. Therefore, perform test PCRs with specific length for your SP.<br>If contamination or wash out is recognized, take the last sample proven to contain phages and free of contamination to start a new PREDCEL iteration round with a fresh culture.
 
−
<strong>Step 8: Plaque assay</strong><br>Perform plaque assays of your PREDCEL samples including inoculation phages and culture samples from <em>step 6</em> (see <em>plaque assay</em> protocol) to calculate phage titers, and to check for washout.
 
−
<strong>Step 9: Check for mutations</strong><br>Pick several plaques to use them for insert amplification via PCR. Sequence your insert with sequencing primers. Mention that mutations taking place while PREDCEL, it may lead to an inefficient primer annealing. Therefore, order several primers and try to find binding regions in conserved parts of your gene that are unlikely to mutate.
 
−
<strong>Troubleshooting</strong>:<br>As phage propagation rate and mutation efficiency usually varies between different gene circuits and phages, but also due to the achieved changes in activity within a PREDCEL run. Thus, you might have to adapt several parameters to achieve a lower selection pressure (if phages get lost over time), a higher selection pressure (only random mutations occur) or changes in mutation rate (too little/ many mutations). The following parameters can be varied:
 
−
<ul>
 
−
<li>the amount of culture volume to be infected and the corresponding flask size</li>
 
−
<li>the time of incubation post infection</li>
 
−
<li>the amount of phage supernatant transferred</li>
 
−
</ul>
 
−
To check for the right parameters, pre-experiments need to be performed for an optimized propagation efficiency.
 
−
You should also prepare several strains of your bacteria carrying APs with different RBS-strengths, origins of replication, and different MPs (MP1/4/6 vary the mutation rate).
 
−
If you have problems with phage propagation due to no or too little starting activity, an initial drift phase without selection pressure might help.
 
−
Another helpful adaption is the usage of a helper-culture. This culture should be used between every or several PREDCEL AP-iteration rounds and must provide geneIII expression, that is only coupled to phage infection. Thereby, all phages that are transferred can reproduce themselves to gain a high phage titer, that might be needed to prevent phage washout. As previous phages showing higher activity propagated faster than those showing no or low activity, they will, as a consequence, achieve higher phage titers while this helper-culture-phase. Thus, helper-culture phase should not extinguish previously achieved selection for beneficial mutations.
 
−
}}|
 
−
}}|table_id-17}}
 
−
{{Heidelberg/accord|Recovery Filter Paper|{{Heidelberg/templateus/Tablebox||{{#tag:html|<h2 id="-recovery-of-plasmids-from-filter-paper">Recovery of plasmids from filter paper</h1>
 
−
<ol>
 
−
<li>Cut out the spot, where the DNA should be, and transfer it into a 1.5 ml reaction tube.
 
−
</li>
 
−
<li>Add 30 µl of dH2O, and mix by pipetting. Incubate for 10 minutes.
 
−
</li>
 
−
<li>Use the DNA solution for fransformation.
 
−
</li>
 
−
</ol>
 
−
}}|
 
−
}}|table_id-18}}
 
−
{{Heidelberg/accord|Restriction Digestion|{{Heidelberg/templateus/Tablebox||{{#tag:html|<h2 id="-restriction-digestion">Restriction digestion</h1>
 
−
<ol>
 
−
<li>Mix the reaction components as follows:</li>
 
−
</ol>
 
−
{{Heidelberg/templateus/Tablebox||{{#tag:html|<table class="table table-bordered mdl-shadow--4dp" XSSCleaned="overflow-x: scroll !important">
 
−
<thead>
 
−
<tr>
 
−
<th>Component</th>
 
−
<th>Volume</th>
 
−
</tr>
 
−
</thead>
 
−
<tbody>
 
−
<tr>
 
−
<td>10X reaction buffer (usually CutSmart Buffer (NEB))</td>
 
−
<td>1 µl</td>
 
−
</tr>
 
−
<tr>
 
−
<td>Restriction enzyme (NEB)</td>
 
−
<td>1 µl</td>
 
−
</tr>
 
−
<tr>
 
−
<td>plasmid DNA</td>
 
−
<td>~ 1 µg</td>
 
−
</tr>
 
−
<tr>
 
−
<td>dH2O</td>
 
−
<td>adjust to 15 µl</td>
 
−
</tr>
 
−
</tbody>
 
−
</table>}}|}}
 
−
<ol>
 
−
<li>Incubate for 1 h at the respective temperature (37 °C for the majority of enzymes).
 
−
</li>
 
−
<li>Heat-inactivate at 85 °C for 20 min if necessary
 
−
</li>
 
−
</ol>
 
−
.4. Separate the DNA fragments via gelelectrophoresis or use them for subsequent ligation<br>.
 
−
}}|
 
−
}}|table_id-19}}
 
  
−
}}
 
−
}}
 
−
}}{{Heidelberg/footer}}
 

Revision as of 17:01, 31 October 2017