Team:Wisconsin-Madison

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      <p class = "classtheoverview"> <strong> The Translational Coupling Cassette: a tool for evaluating the translation of heterologous proteins in <i>Escherichia coli</i>. </strong></p>
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      <p align="left" class = "classtheinlinecontent2"> A powerful method for the production of novel metabolites is the expression of heterologous enzymes in a bacterial host. A common challenge when using non-native genes in metabolic engineering is determining if they are being properly expressed. To address this issue, we have constructed a BioFusion-compatible system for testing the translation of a gene of interest. This system couples the translation of the target gene to a fluorescent reporter gene; fluorescence will only be detected when the target gene is entirely translated. This construct enables synthetic biologists to quickly determine if a gene is being expressed without the need for costly antibodies or analytical instruments (e.g. mass spectrometry). Currently, we are utilizing this cassette to optimize the expression of limonene synthase, an enzyme that catalyzes the production of limonene, a monoterpene with potential as a renewable jet fuel.</p>
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<div id = "divthecontent">
 
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<div id = "divtheprotocol" align="left">
 
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<p class="classtheinlinecontent">
 
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<h1>Alkaline lysis plasmid extraction</h1>
 
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<h2>Solutions</h2>
 
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<h3>Alkaline Lysis Solution 1</h3>
 
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<p class="classtheinlinecontent">
 
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    <li>50 mM glucose</li>
 
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    <li>25 mM Tris-Cl (pH 8.0)</li>
 
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    <li>10 mM EDTA (pH 8.0)</li>
 
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Prepare Solution I from standard stocks in batches of -100 ml, autoclave for 15 minutes at 15 psi (l.05 kg/cm2 ) on liquid cycle, and store at 4 degrees C.
 
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<h3>Alkaline Lysis Solution 2</h3>
 
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    <li>0.2 N NaOH (freshly diluted from a 10 N stock)</li>
 
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    <li>1% (w/v ) SDS</li>
 
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Prepare Solution II fresh and use at room temperature.</p>
 
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<h3>Alkaline Lysis Solution 3</h3>
 
-
 
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    <li>5 M potassium acetate - 60.0 ml</li>
 
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    <li>Glacial acetic acid - 11.5 mL</li>
 
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    <li>H2O - 28.5 ml</li>
 
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The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at 4 degrees C and transfer it to an ice bucket just before use.
 
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</p>
 
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<h2>Protocol</h2>
 
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<p class="classtheinlinecontent">
 
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    <ul>1. Pellet the overnight cultures in a 1.5ml eppendorf tube for 1 min. Spin down 4 to 4.5 ml of culture. (For low copy spin down about 6 ml.</ul>
 
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    <ul>2. Resuspend each pellet in 200ul of Alkaline Lysis Sol I, RnaseA added (final RNaseA concentration should be 100ug/ml) Make sure there are no lumps, homogenized.</ul>
 
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    <ul>3. Add 400ul Alkaline Lysis Sol II. Invert 4-6 times to mix. Do not allow reaction to lyse for more than 5min. Sample should clarify.</ul>
 
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    <ul>4. Add 300ul Alkaline Lysis Sol III. Invert 4-6 times to mix. Sample should have a white precipitate.</ul>
 
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    <ul>5. Add 100ul of chloroform. Do this in a fume hood. Invert 4-6 times to mix.</ul>
 
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    <ul>6. Rest on ice for 5-10min. This step is so that the chloroform does not get too hot in the centrifuges and leak out of the tubes. If you want to skip this step you might consider using less cholorform. I put the tubes at -20 for a couple of minutes.</ul>
 
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    <ul>7. Centrifuge at max. speed (14,000rpm) for 10min.</ul>
 
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    <ul>8. Pipet 750ul of supernatant/aqueous layer into a fresh tube. I do up to 800ul.</ul>
 
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    <ul>9. Add 1/10 volume (75ul) 3M NaOAc, pH 5.2. Flick to mix. 80ul if have 800ul of supernatant.</ul>
 
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    <ul>10. Add 0.7-1.0 volume COLD isopronanol (in freezer). Vortex/Flick to mix. If in a hurry go straight to step 11, otherwise rest on ice for 10-30minutes. I have even let it precipitate overnight at 4C if convenient. 600ul isopropanol. Then I put it at -20C for 5minuetes up to over the weekend if needed.</ul>
 
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    <ul>11. Centrifudge at max. speed for 25min. Most miniprep protocols say to do this at 4C, but I have not noticed decreased yield by centrifuging at room temp.</ul>
 
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    <ul>12. Remove and discard the supernatant. Don’t disturb the pellet. Sometimes I can’t see a pellet and more often than not I still have DNA.</ul>
 
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    <ul>13. Add 1ml of 70% EtOH (at room temp.) Invert 4-6 times to rinse the tube.</ul>
 
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    <ul>14. Centrifuge at max speed for ~5 minutes. Room temp. is fine. Remove and discard the EtOH.</ul>
 
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    <ul>15. Repeat steps 14 and 15 to remove all traces of isopropanol. Pulse spin after removing bulk of final EtOH wash and pipet off remaining EtOH.</ul>
 
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    <ul>16. Air dry the pellet for ~15min (pellet will change from white to clear as it dries). Resuspend desired volume (~30ul) of H2O or EB or T10E1 depending on downstream applications. If you pipet off the EtOH well, then I have done this for as little as 2 min. For fosmids, I usually resuspend the pellet in 20ul water.</ul>
 
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</p>
 
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<h1>Colony PCR</h1>
 
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Colony PCR is an efficient way to screen for the construct with the desired insert. Colony PCR uses primers that attaches on the outside of the cloning site and amplify only the cloning region.<br><br>
 
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<strong style="font-style:italic;">Master Mix</strong><br>
 
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This is for 10ul reactions. The volume below is for one 10ul reaction. If doing more than one, multiply the amount by how many colonies you will be screening, and probably add one extra reaction worth to account for pipetting error.
 
<br>
<br>
<br>
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<div style=" float:left;">
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<p align="left" class="classtheinlinecontent"><strong><span style="font-size:24px"></strong>
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<strong style="font-style:italic;">Reagent Volume</strong><br>
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</p><br />
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<li>Sterile H20 4ul</li>
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<p align="left" class="classtheinlinecontent2"></p>
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<li>GoTaq 5.0ul</li>
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<br />
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<li>FWD primer 0.5ul</li>
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<p align="left" class="classtheinlinecontent"><strong><span style="font-size:24px"></strong>
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<li>REV primer 0.5ul</li>
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<li>In each PCR tube, place 1 colony DNA and 10ul of Master Mix.</li>
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<h3>Thermal Cycler Protocol</h3>
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Step Temperature Time
 
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<ul>1. 95C 3 min</ul>
 
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<ul>2. 95C 30sec</ul>
 
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<ul>3. 55C 30sec</ul>
 
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<ul>4. 72C 3min (depends on length 1min/kb)</ul>
 
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<ul>5. Go to step 2 24x</ul>
 
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<ul>6. 72C 5 min</ul>
 
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<ul>7. 4C Forever</ul>
 
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After the thermal cycler is done, run on a 1% gel at 100V for 30min. Use colonies that display the correct band to make overnight cultures with antibiotic to miniprep the next day for sequencing.
 
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<p align="left" class="classtheinlinecontent2">
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<h1>Digestion for cloning</h1>
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</p><br>
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Sterile Milli-Q Water (20 - x) uL x=amount of reagents used
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Total volume should be 20uL
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<table width="400" border="1">
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    <td>DNA (2000ng/[DNA])</td>
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    <td>_ uL</td>
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  </tr>
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  <tr>
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    <td>BSA* [10X]</td>
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    <td>2 uL</td>
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  </tr>
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  <tr>
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    <td>B4 Buffer</td>
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    <td>2 uL</td>
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  </tr>
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  <tr>
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    <td>Enzyme #1</td>
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    <td>1 uL</td>
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  </tr>
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  <tr>
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    <td>Enzyme #2</td>
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    <td>1 uL</td>
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  </tr>
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  <tr>
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    <td>ddH2O</td>
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    <td>_ uL</td>
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  </tr>
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  <tr>
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    <td><strong>Total</strong></td>
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    <td>20uL</td>
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  </tr>
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</table>
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*BSA should only be added if needed. Check the NEB poster on the -80 to see if it is needed.<br>
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Incubate for 1 hour in the 37 degree water bath<br>
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Notes:<br>
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Keep everything on ice when thawing and mixing reagents. Do Not vortex the master mix; you can gently bump it with your fingers and then spin it down for less than 10 seconds.<br>
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If your DNA [ ] is lower and your volume will exceed 20uL, scale up the amounts of B4 Buffer and BSA to 3uL each and make a 30uL total volume reaction.<br>
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IMPORTANT: Enzymes must be kept in the blue cold box.<br>
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<h1>Dishwashing protocol</h1>
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<ul>1. Bleach / wait / remove stickers</ul>
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<ul>2. Pour down drain</ul>
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<ul>3. Rinse 2-3 times with tap water</ul>
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<ul>4. Add alconox and brush if necessary</ul>
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<ul>5. Rinse 3x with DI, put upside down on a rack</ul>
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<br>
<br>
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<h1>Electroporation</h1>
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</div>
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</div>
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Use the electro-competent cells you just made for transformation.
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ALL OF THIS IS ON ICE
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    <ul>1. 100uL electrocompetent cell aliquots and UV sterilized cuvettes should already be in an ice bucket. (UV means 3 mins in the gel doc on full)</ul>
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    <img src="http://vtb.bme.wisc.edu/image/temp/iGEMFooter2.png" width="960"></div>
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    <ul>2. Label 13mm plastic test tubes beforehand, have 900uL of LB ready to pipette into the cuvette immediately after shocking</ul>
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    <ul>3. Set electroporator machine to bacteria</ul>
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  </center>
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    <ul>4.  If plasmid DNA is ~150 ng/uL use 2.5uL. If 200 ng/uL, use 2uL (~400ng) into 100uL aliquots of electocomp cells</ul>
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    <ul>5. Use a kimwipe to wipe off any condensation from the exterior of the cuvette - important!!</ul>
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    <ul>6. Place the cuvette in the holder in the right orientation, slide in until you hear the click. The two electrodes should touch the sides of the cuvette</ul>
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    <ul>7. Take up 900uL of LB into the pipette tip</ul>
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    <ul>8. Press the SHOCK BUTTON</ul>
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    <ul>9. Immediately, pull out the cuvette and add the 900uL LB directly to the slit inside</ul>
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    <ul>10. Pipette up and down twice very gently. Transfer to the labelled 13mm tube (Don't make bubbles)</ul>
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    <ul>11. Repeat for all samples, INCLUDING YOUR NEGATIVE AND POSITIVE CONTROLS</ul>
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    <ul>12. The time constant reading should be between 3.8 and 5 ms (If it arcs, it is not usable)</ul>
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    <ul>13. Incubate in the gentle shaker (37 degrees and 100rpm) for 1 hour</ul>
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    <ul>14. Plate 100uL aliquots onto appropriate media, leave the remaining 800uL in the tubes to sit on the bench top overnight</ul>
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    <ul>15. For more difficult transformations, pellet 700uL of the culture, pour off supernatant and re-suspend pellet in 100uL of LB. Plate the concentrated 100uL of transformation.</ul>
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    <ul>16. To the remaining 200uL of transformation, add 1mL of LB and let sit on bench top overnight</ul>
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    <ul>17. Incubate all plates in 37oC incubator overnight, inspect for colonies in morning.</ul>
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<br>
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<h1>Ethanol Precipitation</h1>
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<ul>1. 1/10 volume of Sodium Acetate (3 M, pH 5.2).</ul>
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<ul>2. Add 2.5-3.0 X volume (calculated after addition of sodium acetate) of at least 95% (ice-cold) ethanol.</ul>
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<ul>3. Incubate on ice for 15-30 minutes. In case of small DNA fragments (<100bp) or high dilutions overnight incubation gives best results, incubation below 0 °C does not significantly improve efficiency.</ul>
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<ul>4. Centrifuge at > 14,000 x g (max speed) for 10 minutes at room temperature or 0 °C.</ul>
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<ul>5. Remove supernatant being careful not to disturb the DNA pellet, which may be invisible. Remove any drops of liquid that adhere to the walls of the tube. (It is best to save the supernatant from valuable DNA samples until recovery of the precipitated DNA has been verified). </ul>
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<ul>6. Fill tube 1/2 way with 70% Ethanol to rinse and recentrifuge at maximum speed for 2 minutes.</ul>
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<ul>7. Repeat step 5.</ul>
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<ul>8. If necessary, store the open tube on the bench at room temperature just until the last traces of fluid have evaporated.</ul>
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<ul>9. Dissolve pellet (which is often invisible) in desired volume of buffer. (Usually TE [pH between 7.6 and 8.0], or water)</ul> <br>
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NOTE: Make sure to rinse the wall of the tube well with the buffer since up to 50% of the DNA will have been smeared on the wall. This can be done by pushing a bead of fluid over the surface of the wall using a disposable pipette tip.
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<h1>Freezer stock preparation</h1>
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<h3>Freezer Stocks</h3>
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750ul of culture
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250ul of 60% Glycerol Filter Sterilized using equipment in the Pfleger Lab
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Store them in -80C
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Combine in labelled (strain, date, initials) cryotube (lids & tubes in separate blue boxes on center of benches)
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Latest revision as of 20:08, 26 October 2012


The Translational Coupling Cassette: a tool for evaluating the translation of heterologous proteins in Escherichia coli.

A powerful method for the production of novel metabolites is the expression of heterologous enzymes in a bacterial host. A common challenge when using non-native genes in metabolic engineering is determining if they are being properly expressed. To address this issue, we have constructed a BioFusion-compatible system for testing the translation of a gene of interest. This system couples the translation of the target gene to a fluorescent reporter gene; fluorescence will only be detected when the target gene is entirely translated. This construct enables synthetic biologists to quickly determine if a gene is being expressed without the need for costly antibodies or analytical instruments (e.g. mass spectrometry). Currently, we are utilizing this cassette to optimize the expression of limonene synthase, an enzyme that catalyzes the production of limonene, a monoterpene with potential as a renewable jet fuel.









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