Team:Uppsala/Project Notebook

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document.getElementById("tab1").innerHTML = '<h2>Standard iGEM protocols</h2><p>Here we have gathered protocols that most iGEM teams use during their everyday lab work. For example making competent cells and the cloning cycle. Most of our standard protocols originates from a book written with help from previous iGEM Uppsala teams and can therefore not be published here. However we advise you to purchase the book since it is very educational and includes several improved protocols.<br><br>Following protocols were taken from “Synthetic Biology, a lab manual”, chapter 6. Protocols<sup><a href="#reference1">[1]</a></sup>.</p><br><br><ul><li>1. 0.9% NaCl</li><li>2. 50% Glycerol</li><li>3. 1M CaCl2</li><li>4. 10x TBE Buffer</li><li>5. SOB Medium</li><li>6. LB Medium</li><li>7. LB Agar Plates and Addition of Antibiotics</li><li>8. Overnight Cultures with antibiotics, and glycerol stock</li><li>9. Agarose gel electrophoresis</li><li>10. Preparation of competent <i>E. coli</i> cells using CaCl2</li><li>11. Transformation of CaCl2 competent <i>E. coli</i> cells</li><li>12. Bacterial re-streak techniques</li><li>13. Inverse PCR mutagenesis</li><li>14. Digestion with DpnI</li><li>15. Ligation</li><li>16. Colony PCR</li></ul><br><br><p>BioBrick 3A assembly according to NEB protocol, performed with NEB reagents.PCR according to manufacturers protocol. We used NEBs polymerase Q5 for important PCR-reactions and Thermofishers REDTAQ for screening re-streaked colonies.<br><br>All codon optimisation was done for general <i>E. coli</i> using a web tool:<br><a href="http://eu.idtdna.com/CodonOpt">http://eu.idtdna.com/CodonOpt</a></p><h2>Protocols for characterization</h2><p>The first step in preparing a characterisation experiment using IMAC (Immobilized Metal Affinity Chromatography) is to grow liquid cultures overnight. The cells are then lysed by sonifaication</p><h3>Sonication</h3><br><p><b>Day 1</b><br>Grow a culture of the desired constructs in LB 16-20 h (37 degrees Celsius) over night. (Don’t forget to also prepare negative controls, one culture without the construct being assessed.</p><br><br><p><b>Day 2</b><br>Centrifuge the samples for 15 min at 3000 g. Discard the supernatant and resuspend the pellet in 500 µl of 20 mM TRIS buffer. Make sure too much time does not pass between centrifugation and discarding of the supernatant.</p><br><table id="partsT"><tr><th>TRIS Buffer pH 8</th></tr><tr><td>20 mM TRIS</td></tr><tr><td>50 mM NaCl</td></tr></table><br><ul><li>Add lysozyme, the final concentration should be 0,02 mg/ml</li><li>Transfer the samples to 2 mL eppendorf tubes. Lyse the cells by sonication.</li><li>Centrifuge the tubes for 15 min at maximum speed. (transfer to smaller tubes to be able to centrifuge at a higher speed if needed)</li><li>Transfer the supernatant to new tubes</li></ul><h3>IMAC column</h3><p>Our protein was designed with a histidine tag on the N-terminal of the protein, it has affinity towards the nickel ions in the IMAC gel. When the Histidine tag binds to the nickel ions in  the column,it is possible to filter out most of the other proteins that do not have affinity towards the negatively charged gel. Thereby we will hopefully end up with pure colicin Fy. <br><br>Usually IMAC is performed with a pump but since our system is fairly small, gravitation was enough in this experiment. Two columns were used in these experiments. That way it was possible to pour the buffers into the one situated higher up without disrupting the gel in the second column as well as giving a steady flow.<br><br></p><table><tr><td><img src="https://static.igem.org/mediawiki/2014/a/aa/Uppsala-igem2014-Imac2.png"></td><td><i>Figure 1. The IMAC and how it was assembled</i></td></tr></table><p>The end product from the IMAC was collected in 11 tubes for each bacterial strain containing two ml of liquid each. Other eluents in the previous steps were saved in order to be able to see if the protein was eluted in an earlier phase. By using a nanodrop, the protein concentration of each tube was analysed. Analyzing the protein concentration in the tubes by nanodrop resulted in that tube 4 and 6 usually had higher concentration than the other tubes and were therefore taken to the next step.<br><br><b>Charging the column with metal ions</b></p><ul><li>1. Prepare a 0.1 M stock solution of the desired metal ion (Cu2+, Zn2+, Ni2+, Co2+, Fe3+, etc.) in distilled water.</li> <li>2. Wash the column with 5 column volumes (CV) of distilled water.</li><li>3. Apply1 CV of 0.1M metal ion solution to the column.</li><li>4. Wash the column with at least 5 CV of distilled water to remove excess metal ions. Note that the use of buffer instead of water immediately before and after the application of metal ion solution may cause precipitation.</li><li>5. Wash with 5 CV of the buffer that has been chosen for the protein elution, e.g. imidazole elution buffer for competitive elution. Do NOT use EDTA/EGTA regeneration buffer at this stage!</li><li>6. Equilibrate with 10 CV of binding buffer (20 mM IMAC buffer).The column is now ready for use.</li></ul><br><br><ul><li>1. Save a small aliquot of the filtered lysate for further controls. Gently mix your filtered lysate with 1/3 volume of 4x IMAC buffer. Load the lysate on the IMAC gel at the rate of 1 ml/min. Collect the throughput for safety in one batch, label and store.</li> <li>2. Wash the column with 15 CV of 20 mM IMAC buffer, or overnight. Collect again for safety, label and store.</li><li>3. Wash the column with 15 CV of 100 mM IMAC buffer. Collect also this time, label and store.</li><li>4. Elute with 300 mM IMAC buffer and collect the first 30 mL eluent into 1.5 mL Eppendorf tubes.</li><li>5. Identify fractions containing StEH1 by means of the activity assay.</li></ul><h3>SDS-page</h3><p>After having purified the protein with IMAC, the next step is to show that it is there. SDS-page in theory works like a normal gel electrophoresis but for proteins instead of for nucleic acids. Proteins get a negative charge and a linear structure due to the substances SDS, DTT as well as heating them to 95 degrees centigrade for 5 minutes and thereby it is possible to distinguish them by size alone. With a protein ladder it is possible to see what size the other proteins in the gel have.</p><img style="display:block; margin-left: auto; margin-right: auto" src="https://static.igem.org/mediawiki/2014/a/a0/Uppsala-igem2014-SDS.png"><i>Figure 3: SDS-page during the run</i><br><br><p>Make sure you have these things ready before starting:</p><ul><li>4x Seprataion buffer</li><li>8x Stacking buffer</li><li>8x Running buffer</li><li>30% Acrylamidsol</li><li>10% APS</li><li>TEMED</li><li>The samples ready</li><li>Destainer</li><li>1 M DTT</li></ul><b>Day 1</b><p>Cast two 10 % acrylamide gels<br>Caution! Acrylamide is poisonous, always wear gloves, lab coat and safety glasses!</p><i><b>Separation gel</b> (the protocol covers the making of <u>two gels</u>)</i><table id="partsT" style="width: 100%"><tr><th></th><th>12%</th><th><b>10%</b></th><th>8%</th><th>7.5%</th></tr><tr><td>4xSeparationbuffer</td><td>2.5 ml</td><td><b>2.5 ml</b></td><td>2.5 ml</td><td>2.5 ml</td></tr><tr><td>30% 30:08 Acrylamid sol.</td><td>4 ml</td><td><b>3.33 ml</b></td><td>2.7 ml</td><td>2.5 ml</td></tr><tr><td>H<sub>2</sub>O</td><td>3.35 ml</td><td><b>4.02 ml</b></td><td>4.65 ml</td><td>4.85 ml</td></tr><tr><td>10% APS</td><td>50 µl*</td><td><b>100 µl</b></td><td>50 µl*</td><td>50 µl*</td></tr><tr><td>TEMED</td><td>5 µl*</td><td><b>10 µl</b></td><td>5 µl*</td><td>5 µl*</td></tr></table><i>* Alterations might be needed.</i><br><br><i><b>Stacking gel</b> (the protocol is enough for <u>two gels</u>)</i><table id="partsT" style="width: 100%;"><tr><th></th><th>4%</th><th><b>5%</b></th></tr><tr><td>8 x Stacking Buffer</td><td>625 µl</td><td><b>625 µl</b></td></tr><tr><td>30% 30:08 Acrylamid sol.</td><td>650 µl</td><td><b>833 µl</b></td></tr><tr><td>H<sub>2</sub>O</td><td>3.7 ml</td><td><b>3.5 ml</b></td></tr><tr><td>10% APS</td><td>50 µl*</td><td><b>100 µl</b></td></tr><tr><td>TEMED</td><td>5 µl*</td><td><b>10 µl</b></td></tr></table><i>* Alterations might be needed</i><br><br><table id="partsT"><tr><th><b>2x Loading Buffer</b></th><tr><td>125 mM Tris base pH 6.8</td></tr><td>5% SDS</td></tr><td>25 % Glycerol</td><tr><td>0.04% BFB</td></tr></table><br><br><table id="partsT"><tr><th><b>4x Separation Buffer</b></th></tr><tr><td>1.44M Tris base pH 8.8</td></tr><tr><td>0.4% SDS</td></tr></table><br><br><table id="partsT"><tr><th>8x Running Buffer (1L)</th></tr><tr><td>24.2g Tris base</td></tr><tr><td>115.2g Glycin</td></tr><tr><td>8g SDS</td></tr><tr><td>ph 8.3</td></tr></table><br><br><table id="partsT"><tr><th>8x Stacking Buffer</th></tr><tr><td>0.92M Tris base pH 6.8</td></tr><tr><td>0.8% SDS</td></tr></table><br><br><p>Wash the casting equipment carefully with detergent and then with 70% ETOH. Dry with tissues. Put the casting-tray together (use equal pressure when fastening the glass plates). Be sure that the bottoms of the glass plates are equal in height, otherwise the gel will leak...<br><br>Prepare the 10% separation gel according to the protocol and pour it between the glass plates up to 2 cm below the upper edge. Pour n-butanol on top (to get rid of any unwanted air bubbles) and let polymerize for at least 30 min. You need to be quick after adding the APS and TEMED as the gel immediately will begin to polymerize!<br><br>When the separation gel has polymerized (check by carefully tilting the gel), remove the butanol with a piece of filter paper.<br><br> Cast the 5 % stacking gel, be quick after adding APS and TEMED. Don’t forget to add the comb! Make sure there are no bubbles (you can prevent this by adding the comb from one side to the other).<br><br>Prepare Loading Dye by adding 30 µl of 1M DTT to 120 µl 2x Loading Buffer. Add this to your samples and your positive control according to the scheme below.<br><br></p><table id="partsT"><tr><td>20µl of supernatant + 15 µl of Loading Dye (*just enough for one gel!)</td></tr><tr><td>+pellet + 150 µl of Loading Dye (resuspend)</td></tr><tr><td>10 µl Positive Control + 10 µl Tris-buffer + 6 µl Loading Dye</td></tr></table> <br><br><p>Put the gels in the gel tank and add 1x running buffer. Wash the wells with running buffer. Make sure the tank is not leaking.<br><br>Boil the samples in 95 °C for 5 min. Load the samples and the marker on the gel. If you have difficulties loading the pellet because it is too slimy, you can dilute it by adding some running buffer. <br><br>Run the gel on 90-100 V until the dye starts to run out.<br><br>The gel should now be stained with Coomassie Blue. If you would rather continue with the destaining day 2, you can leave the gel in the Coomassie Blue overnight.</p><h3>Project specific</h3><p>There are some protocols that we custom built for our project this year. We summarized all of them under here.</p> <h3>Swarming Plates</h3><p><u>Purpose:</u> To test motility of cells<br><u>General experiment:</u> Special agar plates with a lower concentration of agar. This allow the cells to be able to swim in the plate and colonies a larger area quicker. This can be seen as a “bacterial circle” appearing for motile strains</p><ul><li>1. Create Tryptone Broth by mixing 10 g/L bactotryptone, 5g/L NaCl</li><li>2. Add 2.5 g of agar per liter</li> <li>3. Autoclave to melt and mix agar, and to make sure that the agar-mix is sterile</li><li>4. Let cool to about 50-60 degrees celsius</li><li>5. Add antibiotics to working concentrations, if needed. Swirl flask gently to mix, avoid bubbles.</li><li>6. Let plates cool. Store plates in cold dark environment.</li><li>7. “Dipp” a toothpick of a single colony halfway through the agar. Move the pick up and down to ensure an even spread. Controls should be plated on the same plate.</li><li>8. Seal the lid with “parafilm” and store in room temperature.</li><li>9. Formations of circles should appear after about 2-3 days of incubation</li></ul><h3>Flow Cytometer - FACS</h3><p>To measure the efficiency of promoters we measure individual cell fluorescence with a flow cytometer. All flow cytometer have different protocols and routines, which means that these protocols might not be applicable for your tests with your flow cytometer.<br><u>Model:</u> BDFACS Aria UII<br><br><u>Materials:</u><br></p><ul><li>Flow cytometry tubes, adjusted for your machine</li><li>PBS 2mL x (number of strains)</li><li>Growth Medium, e.g. LB with appropriate selective marker to ensure no loss of plasmid</li><li>Agar plates with selective marker</li><li>Inoculation-loop 3x(number of strains</li><li>Falcon tubes 4x(number of strains to test)</li></ul><br><br><p><u>2-days prior to measurement:</u><br>Re-streak a single colony of all of your strains. Also re-streak a bacteria of the same strain that is non-fluorescent.<br><br><u>1-day prior to measurement:</u><br>Create overnight cultures for four clones of each strain, let grow to stationary phase (~18h).<br><br><u>2-hours prior to measurement:</u><br>Mix 500microL of PBS with 2-10microL overnight culture depending on cell concentrations for each overnight. You don’t want too much cells, as this might lead to a fluorescence outside the spectra, but neither do you want to few, since this will result in a high variance between samples. Let the cells incubate in PBS for at least 1 hour.<br><br><u>Measurement:</u><br>Start with measuring the highest expression cells. Make sure to measure noise with water first and vortex each tube for a couple of seconds before measurements. If the measurement was “MAXed out” adjust the voltage over the photomultiplier tube until you got a good value. Then proceed with measuring each tube of PBS. If you get a maxed out value during the measurement you need to restart, since the machine measures in relative units. This means that all samples must be measured with the same wave length.<br><br>When analysing data look for clear peaks. When no clear peak can be found the limit of fluorescence can be adjusted by setting the limit so that only 0.5% of the non-fluorescent cells from your control is included. This way you ensure that your limit is not too far away, meaning that you would miss a lot of fluorescent cells, but not too close and thus does not include too many non-fluorescent cells.<br><br>Mean of fluorescence is often taken as “median” or “geometric mean” of the peaks. You will always get a high cell to cell variation since there are cells in different stages of their cell cycle</p><h3>Growth Test</h3><p><u>Purpose:</u> Measure how different conditions affect growth<br><u>General idea:</u> By growing the cells in different conditions and taking several measurements, the growth can be followed.<br><br><u>Materials:</u></p><ul><li>Overnight cultures of testing strains</li><li>Shaker in optimal growth temperature(37 for <i>E. coli</i>)</li><li>Spectrophotometer</li><li>Growth medium (e.g. LB)</li></ul><ul class="reference"><li><a id="reference1">[1]</a>Liljeruhm J, Gullberg E, Forster AC. 2014. Synthetic biology: a lab manual. 1st edition. World Scientific, Singapore.</li>';
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document.getElementById("tab1").innerHTML = '<h2>Standard iGEM protocols</h2><p>Here we have gathered protocols that most iGEM teams use during their everyday lab work. For example making competent cells and the cloning cycle. Most of our standard protocols originates from a book written with help from previous iGEM Uppsala teams and can therefore not be published here. However we advise you to purchase the book since it is very educational and includes several improved protocols.<br><br>Following protocols were taken from “Synthetic Biology, a lab manual”, chapter 6. Protocols<sup><a href="#reference1">[1]</a></sup>.</p><br><br><ul><li>1. 0.9% NaCl</li><li>2. 50% Glycerol</li><li>3. 1M CaCl2</li><li>4. 10x TBE Buffer</li><li>5. SOB Medium</li><li>6. LB Medium</li><li>7. LB Agar Plates and Addition of Antibiotics</li><li>8. Overnight Cultures with antibiotics, and glycerol stock</li><li>9. Agarose gel electrophoresis</li><li>10. Preparation of competent <i>E. coli</i> cells using CaCl2</li><li>11. Transformation of CaCl2 competent <i>E. coli</i> cells</li><li>12. Bacterial re-streak techniques</li><li>13. Inverse PCR mutagenesis</li><li>14. Digestion with DpnI</li><li>15. Ligation</li><li>16. Colony PCR</li></ul><br><br><p>BioBrick 3A assembly according to NEB protocol, performed with NEB reagents.PCR according to manufacturers protocol. We used NEBs polymerase Q5 for important PCR-reactions and Thermofishers REDTAQ for screening re-streaked colonies.<br><br>All codon optimisation was done for general <i>E. coli</i> using a web tool:<br><a href="http://eu.idtdna.com/CodonOpt">http://eu.idtdna.com/CodonOpt</a></p><h2>Protocols for characterization</h2><p>The first step in preparing a characterisation experiment using IMAC (Immobilized Metal Affinity Chromatography) is to grow liquid cultures overnight. The cells are then lysed by sonifaication</p><h3>Sonication</h3><br><p><b>Day 1</b><br>Grow a culture of the desired constructs in LB 16-20 h (37° C) over night. (Don’t forget to also prepare negative controls, one culture without the construct being assessed.</p><br><br><p><b>Day 2</b><br>Centrifuge the samples for 15 min at 3000 g. Discard the supernatant and resuspend the pellet in 500 µl of 20 mM TRIS buffer. Make sure too much time does not pass between centrifugation and discarding of the supernatant.</p><br><table id="partsT"><tr><th>TRIS Buffer pH 8</th></tr><tr><td>20 mM TRIS</td></tr><tr><td>50 mM NaCl</td></tr></table><br><ul><li>Add lysozyme, the final concentration should be 0,02 mg/ml</li><li>Transfer the samples to 2 mL eppendorf tubes. Lyse the cells by sonication.</li><li>Centrifuge the tubes for 15 min at maximum speed. (transfer to smaller tubes to be able to centrifuge at a higher speed if needed)</li><li>Transfer the supernatant to new tubes</li></ul><h3>IMAC column</h3><p>Our protein was designed with a histidine tag on the N-terminal of the protein, it has affinity towards the nickel ions in the IMAC gel. When the Histidine tag binds to the nickel ions in  the column,it is possible to filter out most of the other proteins that do not have affinity towards the negatively charged gel. Thereby we will hopefully end up with pure colicin Fy. <br><br>Usually IMAC is performed with a pump but since our system is fairly small, gravitation was enough in this experiment. Two columns were used in these experiments. That way it was possible to pour the buffers into the one situated higher up without disrupting the gel in the second column as well as giving a steady flow.<br><br></p><table><tr><td><img src="https://static.igem.org/mediawiki/2014/a/aa/Uppsala-igem2014-Imac2.png"></td><td><i>Figure 1. The IMAC and how it was assembled</i></td></tr></table><p>The end product from the IMAC was collected in 11 tubes for each bacterial strain containing two ml of liquid each. Other eluents in the previous steps were saved in order to be able to see if the protein was eluted in an earlier phase. By using a nanodrop, the protein concentration of each tube was analysed. Analyzing the protein concentration in the tubes by nanodrop resulted in that tube 4 and 6 usually had higher concentration than the other tubes and were therefore taken to the next step.<br><br><b>Charging the column with metal ions</b></p><ul><li>1. Prepare a 0.1 M stock solution of the desired metal ion (Cu2+, Zn2+, Ni2+, Co2+, Fe3+, etc.) in distilled water.</li> <li>2. Wash the column with 5 column volumes (CV) of distilled water.</li><li>3. Apply1 CV of 0.1M metal ion solution to the column.</li><li>4. Wash the column with at least 5 CV of distilled water to remove excess metal ions. Note that the use of buffer instead of water immediately before and after the application of metal ion solution may cause precipitation.</li><li>5. Wash with 5 CV of the buffer that has been chosen for the protein elution, e.g. imidazole elution buffer for competitive elution. Do NOT use EDTA/EGTA regeneration buffer at this stage!</li><li>6. Equilibrate with 10 CV of binding buffer (20 mM IMAC buffer).The column is now ready for use.</li></ul><br><br><ul><li>1. Save a small aliquot of the filtered lysate for further controls. Gently mix your filtered lysate with 1/3 volume of 4x IMAC buffer. Load the lysate on the IMAC gel at the rate of 1 ml/min. Collect the throughput for safety in one batch, label and store.</li> <li>2. Wash the column with 15 CV of 20 mM IMAC buffer, or overnight. Collect again for safety, label and store.</li><li>3. Wash the column with 15 CV of 100 mM IMAC buffer. Collect also this time, label and store.</li><li>4. Elute with 300 mM IMAC buffer and collect the first 30 mL eluent into 1.5 mL Eppendorf tubes.</li><li>5. Identify fractions containing StEH1 by means of the activity assay.</li></ul><h3>SDS-page</h3><p>After having purified the protein with IMAC, the next step is to show that it is there. SDS-page in theory works like a normal gel electrophoresis but for proteins instead of for nucleic acids. Proteins get a negative charge and a linear structure due to the substances SDS, DTT as well as heating them to 95 degrees centigrade for 5 minutes and thereby it is possible to distinguish them by size alone. With a protein ladder it is possible to see what size the other proteins in the gel have.</p><img style="display:block; margin-left: auto; margin-right: auto" src="https://static.igem.org/mediawiki/2014/a/a0/Uppsala-igem2014-SDS.png"><i>Figure 3: SDS-page during the run</i><br><br><p>Make sure you have these things ready before starting:</p><ul><li>4x Seprataion buffer</li><li>8x Stacking buffer</li><li>8x Running buffer</li><li>30% Acrylamidsol</li><li>10% APS</li><li>TEMED</li><li>The samples ready</li><li>Destainer</li><li>1 M DTT</li></ul><b>Day 1</b><p>Cast two 10 % acrylamide gels<br>Caution! Acrylamide is poisonous, always wear gloves, lab coat and safety glasses!</p><i><b>Separation gel</b> (the protocol covers the making of <u>two gels</u>)</i><table id="partsT" style="width: 100%"><tr><th></th><th>12%</th><th><b>10%</b></th><th>8%</th><th>7.5%</th></tr><tr><td>4xSeparationbuffer</td><td>2.5 ml</td><td><b>2.5 ml</b></td><td>2.5 ml</td><td>2.5 ml</td></tr><tr><td>30% 30:08 Acrylamid sol.</td><td>4 ml</td><td><b>3.33 ml</b></td><td>2.7 ml</td><td>2.5 ml</td></tr><tr><td>H<sub>2</sub>O</td><td>3.35 ml</td><td><b>4.02 ml</b></td><td>4.65 ml</td><td>4.85 ml</td></tr><tr><td>10% APS</td><td>50 µl*</td><td><b>100 µl</b></td><td>50 µl*</td><td>50 µl*</td></tr><tr><td>TEMED</td><td>5 µl*</td><td><b>10 µl</b></td><td>5 µl*</td><td>5 µl*</td></tr></table><i>* Alterations might be needed.</i><br><br><i><b>Stacking gel</b> (the protocol is enough for <u>two gels</u>)</i><table id="partsT" style="width: 100%;"><tr><th></th><th>4%</th><th><b>5%</b></th></tr><tr><td>8 x Stacking Buffer</td><td>625 µl</td><td><b>625 µl</b></td></tr><tr><td>30% 30:08 Acrylamid sol.</td><td>650 µl</td><td><b>833 µl</b></td></tr><tr><td>H<sub>2</sub>O</td><td>3.7 ml</td><td><b>3.5 ml</b></td></tr><tr><td>10% APS</td><td>50 µl*</td><td><b>100 µl</b></td></tr><tr><td>TEMED</td><td>5 µl*</td><td><b>10 µl</b></td></tr></table><i>* Alterations might be needed</i><br><br><table id="partsT"><tr><th><b>2x Loading Buffer</b></th><tr><td>125 mM Tris base pH 6.8</td></tr><td>5% SDS</td></tr><td>25 % Glycerol</td><tr><td>0.04% BFB</td></tr></table><br><br><table id="partsT"><tr><th><b>4x Separation Buffer</b></th></tr><tr><td>1.44M Tris base pH 8.8</td></tr><tr><td>0.4% SDS</td></tr></table><br><br><table id="partsT"><tr><th>8x Running Buffer (1L)</th></tr><tr><td>24.2g Tris base</td></tr><tr><td>115.2g Glycin</td></tr><tr><td>8g SDS</td></tr><tr><td>ph 8.3</td></tr></table><br><br><table id="partsT"><tr><th>8x Stacking Buffer</th></tr><tr><td>0.92M Tris base pH 6.8</td></tr><tr><td>0.8% SDS</td></tr></table><br><br><p>Wash the casting equipment carefully with detergent and then with 70% ETOH. Dry with tissues. Put the casting-tray together (use equal pressure when fastening the glass plates). Be sure that the bottoms of the glass plates are equal in height, otherwise the gel will leak...<br><br>Prepare the 10% separation gel according to the protocol and pour it between the glass plates up to 2 cm below the upper edge. Pour n-butanol on top (to get rid of any unwanted air bubbles) and let polymerize for at least 30 min. You need to be quick after adding the APS and TEMED as the gel immediately will begin to polymerize!<br><br>When the separation gel has polymerized (check by carefully tilting the gel), remove the butanol with a piece of filter paper.<br><br> Cast the 5 % stacking gel, be quick after adding APS and TEMED. Don’t forget to add the comb! Make sure there are no bubbles (you can prevent this by adding the comb from one side to the other).<br><br>Prepare Loading Dye by adding 30 µl of 1M DTT to 120 µl 2x Loading Buffer. Add this to your samples and your positive control according to the scheme below.<br><br></p><table id="partsT"><tr><td>20µl of supernatant + 15 µl of Loading Dye (*just enough for one gel!)</td></tr><tr><td>+pellet + 150 µl of Loading Dye (resuspend)</td></tr><tr><td>10 µl Positive Control + 10 µl Tris-buffer + 6 µl Loading Dye</td></tr></table> <br><br><p>Put the gels in the gel tank and add 1x running buffer. Wash the wells with running buffer. Make sure the tank is not leaking.<br><br>Boil the samples in 95 °C for 5 min. Load the samples and the marker on the gel. If you have difficulties loading the pellet because it is too slimy, you can dilute it by adding some running buffer. <br><br>Run the gel on 90-100 V until the dye starts to run out.<br><br>The gel should now be stained with Coomassie Blue. If you would rather continue with the destaining day 2, you can leave the gel in the Coomassie Blue overnight.</p><h3>Project specific</h3><p>There are some protocols that we custom built for our project this year. We summarized all of them under here.</p> <h3>Swarming Plates</h3><p><u>Purpose:</u> To test motility of cells<br><u>General experiment:</u> Special agar plates with a lower concentration of agar. This allow the cells to be able to swim in the plate and colonies a larger area quicker. This can be seen as a “bacterial circle” appearing for motile strains</p><ul><li>1. Create Tryptone Broth by mixing 10 g/L bactotryptone, 5g/L NaCl</li><li>2. Add 2.5 g of agar per liter</li> <li>3. Autoclave to melt and mix agar, and to make sure that the agar-mix is sterile</li><li>4. Let cool to about 50-60 degrees celsius</li><li>5. Add antibiotics to working concentrations, if needed. Swirl flask gently to mix, avoid bubbles.</li><li>6. Let plates cool. Store plates in cold dark environment.</li><li>7. “Dipp” a toothpick of a single colony halfway through the agar. Move the pick up and down to ensure an even spread. Controls should be plated on the same plate.</li><li>8. Seal the lid with “parafilm” and store in room temperature.</li><li>9. Formations of circles should appear after about 2-3 days of incubation</li></ul><h3>Flow Cytometer - FACS</h3><p>To measure the efficiency of promoters we measure individual cell fluorescence with a flow cytometer. All flow cytometer have different protocols and routines, which means that these protocols might not be applicable for your tests with your flow cytometer.<br><u>Model:</u> BDFACS Aria UII<br><br><u>Materials:</u><br></p><ul><li>Flow cytometry tubes, adjusted for your machine</li><li>PBS 2mL x (number of strains)</li><li>Growth Medium, e.g. LB with appropriate selective marker to ensure no loss of plasmid</li><li>Agar plates with selective marker</li><li>Inoculation-loop 3x(number of strains</li><li>Falcon tubes 4x(number of strains to test)</li></ul><br><br><p><u>2-days prior to measurement:</u><br>Re-streak a single colony of all of your strains. Also re-streak a bacteria of the same strain that is non-fluorescent.<br><br><u>1-day prior to measurement:</u><br>Create overnight cultures for four clones of each strain, let grow to stationary phase (~18h).<br><br><u>2-hours prior to measurement:</u><br>Mix 500microL of PBS with 2-10microL overnight culture depending on cell concentrations for each overnight. You don’t want too much cells, as this might lead to a fluorescence outside the spectra, but neither do you want to few, since this will result in a high variance between samples. Let the cells incubate in PBS for at least 1 hour.<br><br><u>Measurement:</u><br>Start with measuring the highest expression cells. Make sure to measure noise with water first and vortex each tube for a couple of seconds before measurements. If the measurement was “MAXed out” adjust the voltage over the photomultiplier tube until you got a good value. Then proceed with measuring each tube of PBS. If you get a maxed out value during the measurement you need to restart, since the machine measures in relative units. This means that all samples must be measured with the same wave length.<br><br>When analysing data look for clear peaks. When no clear peak can be found the limit of fluorescence can be adjusted by setting the limit so that only 0.5% of the non-fluorescent cells from your control is included. This way you ensure that your limit is not too far away, meaning that you would miss a lot of fluorescent cells, but not too close and thus does not include too many non-fluorescent cells.<br><br>Mean of fluorescence is often taken as “median” or “geometric mean” of the peaks. You will always get a high cell to cell variation since there are cells in different stages of their cell cycle</p><h3>Growth Test</h3><p><u>Purpose:</u> Measure how different conditions affect growth<br><u>General idea:</u> By growing the cells in different conditions and taking several measurements, the growth can be followed.<br><br><u>Materials:</u></p><ul><li>Overnight cultures of testing strains</li><li>Shaker in optimal growth temperature(37° C for <i>E. coli</i>)</li><li>Spectrophotometer</li><li>Growth medium (e.g. LB)</li></ul><ul class="reference"><li><a id="reference1">[1]</a>Liljeruhm J, Gullberg E, Forster AC. 2014. Synthetic biology: a lab manual. 1st edition. World Scientific, Singapore.</li>';
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