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| - | <html>
| + | <p>Run a <a href="#protocol6">DNA gel</a> using the purified DNA collected yesterday to check DNA is pure. </p> |
| - | <head>
| + | |
| - |
| + | |
| - | </head>
| + | |
| - | <body>
| + | |
| - | <h1 class="subPageTitle">Wet Lab Journal</h1>
| + | |
| - | <div class="gap5px"></div>
| + | |
| - | | + | |
| - | <div class="mainPage">
| + | |
| - | <div class="pageSection1">
| + | |
| - | <div class="sectionHeading">
| + | |
| - | <h1> Week 1</h1>
| + | |
| - | <h5>Monday 23<sup>rd</sup> June</h5>
| + | |
| - | | + | |
| - | <p >Make up 100ml of <a href="#protocol1">LB Broth</a> and 100ml <a href="#protocol2">LB Agar</a>.</p> | + | |
| - | | + | |
| - | <p>Make an <a href="#protocol3">overnight</a> starter culture of <a href="#biobrick1">BBa_K258008</a> to test sterile technique.</p>
| + | |
| - | | + | |
| - | <h5>Tuesday 24<sup>th</sup> June</h5>
| + | |
| - | | + | |
| - | <p>Generate <a href="#protocol4">Chemically competent</a> E.Coli.</p>
| + | |
| - | | + | |
| - | <ol>
| + | |
| - | <li>Make up 8 flasks of 100ml starter culture.</li>
| + | |
| - | <li>4 flasks (A-D) were checked each time to take the OD600 readings.</li>
| + | |
| - | <li>
| + | |
| - | OD600 readings to show when the cells are in exponential growth phase and so ready to be made competent.
| + | |
| - | <table>
| + | |
| - | <tr>
| + | |
| - | <th></th>
| + | |
| - | <th>A</th>
| + | |
| - | <th>B</th>
| + | |
| - | <th>C</th>
| + | |
| - | <th>D</th>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td>1.25h</td>
| + | |
| - | <td>0.077</td>
| + | |
| - | <td>0.078</td>
| + | |
| - | <td>0.082</td>
| + | |
| - | <td>0.096</td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td>3.25h</td>
| + | |
| - | <td>0.567</td>
| + | |
| - | <td>0.674</td>
| + | |
| - | <td>0.714</td>
| + | |
| - | <td>0.809</td>
| + | |
| - | </tr>
| + | |
| - | </table>
| + | |
| - | </li>
| + | |
| - | <li>Aliquot into 24 falcon tubes. The cells will need to be spun down in 2 rounds.</li>
| + | |
| - | <li>72 eppendorf tubes will be produced, labelled ch. comp, dh5alpha.</li>
| + | |
| - | <li>Store at -80°C.</li>
| + | |
| - | </ol>
| + | |
| - | | + | |
| - | <h1> Week 2 </h1>
| + | |
| - | <h5>Monday 30<sup>th</sup> June</h5>
| + | |
| - | | + | |
| - | Make 5mL <a href="#protocol3">overnight</a> starter cultures of the following;
| + | |
| - | <br><br><br><table style="width:50%; color:#696969">
| + | |
| - | <tr>
| + | |
| - | <th>Tube</th>
| + | |
| - | <th>BioBrick</th>
| + | |
| - | <th>Antibiotic</th></tr>
| + | |
| - | <tr><td>1</td> <td><a href="#biobrick1">BBa_K258008</a></td> <td>Chloramphenicol</td></tr>
| + | |
| - | <tr><td>2</td> <td><a href="#biobrick2">BBa_K215107</a></td> <td>Tetracycline</td></tr>
| + | |
| - | <tr><td>3</td> <td><a href="#biobrick3">BBa_K861061</a></td> <td>Chloramphenicol</td></tr>
| + | |
| - | <tr><td>4</td> <td><a href="#biobrick4">BBa_K215002</a></td> <td>Ampicillin</td></tr>
| + | |
| - | <tr><td>5</td> <td><a href="#biobrick5">BBa_K1149021</a></td> <td>Chloramphenicol</td></tr>
| + | |
| - | <tr><td>6</td> <td><a href="#biobrick6">BBa_K861060</a></td> <td>Chloramphenicol</td></tr></table>
| + | |
| - | | + | |
| - | <h4><p class="tab">5μl of an appropriate antibiotic was added to each culture to allow selection for cells containing biobrick plasmids.</p></h4>
| + | |
| - | | + | |
| - | | + | |
| - | <h5>Tuesday 1<sup>st</sup> July</h5>
| + | |
| - | | + | |
| - | Conduct 6 <a href="#protocol5">mini-preps</a>, one for each of the biobrick cultures, to produce 6 Eppendorfs containing purified DNA.
| + | |
| - | | + | |
| - | <h5>Wednesday 2<sup>nd</sup> July</h5>
| + | |
| - | | + | |
| - | <br>Run a <a href="#protocol6">DNA gel</a> using the purified DNA collected yesterday to check DNA is pure. | + | |
| - | <Br><h4><p class="tab">Use an 8 well comb to produce the gel.
| + | |
| - | <br>The table below shows the placement of each DNA sample within the Gel.
| + | |
| - | <br><br>
| + | |
| - | <table style="width:50%; color:#696969"><tr><th>Well</th> <th>Biobrick</th> <th>Part Function</th>
| + | |
| - | <tr><td>1</td> <td>1kb gene ruler</td><td></td></tr>
| + | |
| - | <tr><td>2</td> <td><a href="#biobrick5">BBa_K1149021</a></td> <td>Promoter, RBS, Lipase</td></tr>
| + | |
| - | <tr><td>3</td> <td><a href="#biobrick1">BBa_K258008</a></td> <td>ABC transporter</td></tr>
| + | |
| - | <tr><td>4</td> <td><a href="#biobrick6">BBa_K861060</a></td> <td>pFadR promoter</td></tr>
| + | |
| - | <tr><td>5</td> <td><a href="#biobrick3">BBa_K861061</a></td> <td>pFadR Promoter + RFP</td></tr>
| + | |
| - | <tr><td>6</td> <td><a href="#biobrick2">BBa_K215107</a></td> <td>ABC transporter (strong)</td></tr>
| + | |
| - | <tr><td>7</td> <td><a href="#biobrick4">BBa_K215002</a></td> <td>pLAC +RBS +secretion signal + streptavidin binding tag</td></tr></table>
| + | |
| - | | + | |
| - | <br><br>The gel was run at 100v for 45 minutes (due to time constrains).
| + | |
| - | Below is the image taken of the Gel on the Gel dock.</h4></p>
| + | |
| - | | + | |
| - | 4 LB agar <a href="#protocol7">plates</a> were made, 2 ampicillin (100 μl) and 2 chloramphenicol (25 μl).
| + | |
| - | | + | |
| - | <br><br><a href="#protocol8">Nanodrop</a> the DNA gained from the Mini-prep to generate concentrations of extracted DNA for all 6 biobricks.
| + | |
| - | | + | |
| - | <br><h4><p class="tab">Results are shown below;
| + | |
| - | | + | |
| - | </p></h4>
| + | |
| - | <table style="width:50%; color:#696969"><tr>
| + | |
| - | <tr> <th>Biobrick</th> <th>Concentration (ng/μl)</th></tr>
| + | |
| - | <tr> <td><a href="#biobrick5">BBa_K1149021</a></td> <td>115.0</td></tr>
| + | |
| - | <tr> <td><a href="#biobrick1">BBa_K258008</a></td> <td>075.9</td></tr>
| + | |
| - | <tr> <td><a href="#biobrick6">BBa_K861060</a></td> <td>100.3</td></tr>
| + | |
| - | <tr> <td><a href="#biobrick3">BBa_K861061</a></td> <td>130.7</td></tr>
| + | |
| - | <tr> <td><a href="#biobrick2">BBa_K215107</a></td> <td>043.3</td></tr>
| + | |
| - | <tr> <td><a href="#biobrick4">BBa_K215002</a></td> <td>125.0</td></tr></table>
| + | |
| - | | + | |
| - | | + | |
| - | <br><a href="#protocol9">Transform</a> cells to confirm competency
| + | |
| - | | + | |
| - | <br><h4><p class="tab">For this transformation We used the BioBricks BBa_K258008 and BBa_K215002 to check if the cells were competent.
| + | |
| - | <br>Calculations are shown below;
| + | |
| - | | + | |
| - | <br><br>BBa_K258008
| + | |
| - | <br>75.9ng x X = 10ng x 100μl
| + | |
| - | <br>X = 13.2μl
| + | |
| - | | + | |
| - | <br><br>BBa_K215002
| + | |
| - | <br>125.0ng x X = 10ng x 100μl
| + | |
| - | <br>X = 8.0μl</p></h4>
| + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | <h5>Friday 4<sup>th</sup> July</h5>
| + | |
| - | | + | |
| - | The linearised TliA and KerUS synthesised DNA is cloned into a pJet1.2 plasmid using <a href="#protocol10">blunt end ligation</a>.
| + | |
| - | | + | |
| - | <br><br>This ligation mix was then used to <a href="#protocol9">transform</a> 3 eppendorfs of chemically competant cells (made on tuesday 25th);
| + | |
| - | | + | |
| - | <h4><p class="tab"><li>Into eppendorf 1, pipette 5μl of the ligation mix containing TliA DNA.
| + | |
| - | <br><li>Into eppendorf 2, pipette 5μl of the ligation mix containing KerUs DNA.
| + | |
| - | <br><li>Into eppendorf 3, pipette 5μl of dH<sub>2</sub>O</p></h4>
| + | |
| - | | + | |
| - | <h4><p class="tab"><br>Incubated for 45 minutes due to time constraints.
| + | |
| - | <Br>Constructs plated on ampicillin LB agar plates to select for the correct colonies.
| + | |
| - | <br>Plates were checked on saturday AM and positive colonies were observed</p></h4>
| + | |
| - | | + | |
| - | | + | |
| - | <h1> Week 3 </h1>
| + | |
| - | <h5>Monday 7<sup>th</sup> July</h5>
| + | |
| - | | + | |
| - | Make 200ml <a href="#protocol2">LB Agar</a>and 200ml <a href="#protocol1">LB Broth</a>.
| + | |
| - | | + | |
| - | | + | |
| - | <br><br>Make <a href="#protocol3">overnight</a> cultures of positive colonies of pJet 1.2 with <a href="#biobrickL">TliA</a> and <a href="#biobrickK">KerUS</a>.
| + | |
| - | <br><br>Make <a href="#protocol3">overnight</a> cultures of <a href="#cell1">E. coli MC1000</a>.
| + | |
| - | | + | |
| - | <h5>Tuesday 8<sup>th</sup> July</h5>
| + | |
| - | | + | |
| - | <a href="#protocol5">Miniprep</a> the <a href="#biobrickL">Tlia</a> and <a href="#biobrickK">KerUS</a> cultures to produce purified plasmid DNA.
| + | |
| - | | + | |
| - | <br><br><a href="#protocol8">Nanodrop</a> the purified DNA to determine the concentration of DNA.
| + | |
| - | <h4><p class="tab">
| + | |
| - | TliA - 5.2ng/μl
| + | |
| - | <br>KerUS - 3.4ng/μl </p></h4>
| + | |
| - | | + | |
| - | Produce <a href="#protocol14">glycerol stocks</a> of the Tlia and KerUS cells for storage (retain only if insert confirmed).
| + | |
| - | | + | |
| - | <br><br>Make <a href="#protocol4">chemically competent</a> MC1000.
| + | |
| - | <h4><p class="tab">Measure the OD600 of the MC1000 cells using spectrophotometer
| + | |
| - | <br><br>Flask 1 - 0.803
| + | |
| - | <br>Flask 2 - 0.624
| + | |
| - | <br>Blank - 0.066
| + | |
| - | <br><br>Decant the flasks of MC1000 into 50ml falcon tubes. We have 4 tubes of full MC1000, the 5th one is 21ml MC1000 and 9ml water, the 6th one is all water -to ensure the centrifuge is balanced (weight checked too).</p></h4>
| + | |
| - | | + | |
| - | Make 10ml <a href="#protocol3">overnight</a> cultures of pJet 1.2 containing <a href="#biobrickL">TliA</a> or <a href="#biobrickK">KerUS</a>
| + | |
| - | | + | |
| - | <h5>Wednesday 9<sup>th</sup> July</h5>
| + | |
| - | | + | |
| - | Repeat yesterdays <a href="#protocol5">miniprep</a> as it didn't yield enough DNA.
| + | |
| - | <br><h4><p class="tab">Began with two 10ml starter cultures, one of pJet 1.2 TliA and one of pJet 1.2 KerUS.
| + | |
| - | <br>Decanted by hand into falcon tubes to give four 5ml cultures.</p></h4>
| + | |
| - | | + | |
| - | A <a href="#protocol5">Restriction Digest</a> was carried out on the Tlia and KerUS minipreps, as well as the FadR vector that was miniprepped last week.
| + | |
| - | <Br>TliA and KerUS were cut with xbaI and pstI, whilst FadR was cut with speI and pstI.
| + | |
| - | | + | |
| - | <h5>Thursday 10<sup>th</sup> July</h5>
| + | |
| - | | + | |
| - | <a href="#protocol6">Run</a> Vector, TliA and KerUS DNA on a gel to ensure it has been cut correctly. Once confirmed the DNA was then <a href="#protocol18">extracted</a> from the gel.
| + | |
| - | | + | |
| - | <h4><p class="tab">Add 12.5μl 5X loading buffer to the DNA samples (50μl each) - total of 62.5μl in each eppendorf tube.
| + | |
| - | <br>Load each sample, using two wells each if necessary.
| + | |
| - | <br>Run at 100V for 60 minutes.</p></h4>
| + | |
| - | | + | |
| - | Visualise the gel.
| + | |
| - | <h4><p class="tab"> The bands for TliA and Vector DNA were the correct size.
| + | |
| - | <br> The band for KerUS showed that the DNA had been cut incorrectly.</p></h4>
| + | |
| - | | + | |
| - | The two samples of DNA that have been cut correctly are now extracted from the gel.
| + | |
| - | <br><br>The gel extraction was then nanodropped to determine DNA concentration.
| + | |
| - | <h4><p class="tab">
| + | |
| - | | + | |
| - | | + | |
| - | Vector: 14.3ng/μL
| + | |
| - | <br>Tlia: 190.9ng/μL</p></h4>
| + | |
| - | | + | |
| - | A <a href="#protocol12">ligation reaction</a> was carried out using the following amounts of reactants;
| + | |
| - | | + | |
| - | <h4><p class="tab">3.5 μl vector
| + | |
| - | <br>0.2 μl lipase
| + | |
| - | <Br>2 μl buffer
| + | |
| - | <br>1 μl ligase
| + | |
| - | <Br>13.3 μl dH2O
| + | |
| - | <Br><br>leave for 10 minutes at room temperature</p></h4>
| + | |
| - | | + | |
| - | Add 5μl ligation reaction mix to 200μl DH5α and <a href="#protocol9">transform</a>.
| + | |
| - | | + | |
| - | <br><Br>NOTE: did not work -no positve colonies following antibiotic resistance selection on an LB agar plate
| + | |
| - | <div style="border-bottom:1px solid gold;"></div>
| + | |
| - | | + | |
| - | <Br>We also did some work to help us characterise the pFadR promoter.
| + | |
| - | <H4><p class="tab">
| + | |
| - | Due to a mistake over biobrick numbers, our overnight contained pFadR without RFP, and so we used the day to test our experimental procedures.</p></h4>
| + | |
| - | | + | |
| - | <a href="#protocol15">Day cultures</a> were made.
| + | |
| - | <h4><p class="tab">1 x 5ml of empty DH5α
| + | |
| - | <br>3 x 5ml of DH5α containing <a href="#biobrick6">BBa_K861060</a>
| + | |
| - | <Br>Day cultures were placed in the incubator at 37°C, 170rpm for 3 hours</p></h4>
| + | |
| - | | + | |
| - | <a href="#protocol20">Lipid stocks</a> were made using Triton to solubilise.
| + | |
| - | | + | |
| - | <br><Br>12 samples were made to be assayed;
| + | |
| - | <h4><p class="tab">1: 5ml DH5α + 0% lipid
| + | |
| - | <Br>2: 5ml DH5α + 1.0% olive oil
| + | |
| - | <br>3: 5ml DH5α + 1.0% oleic acid
| + | |
| - | <br>4: 5ml DH5α + BBa_K861060 + 0% lipid
| + | |
| - | <br>5: 5ml DH5α + BBa_K861060 + 0.1% olive oil
| + | |
| - | <br>6: 5ml DH5α + BBa_K861060 + 1.0% olive oil
| + | |
| - | <br>7: 5ml DH5α + BBa_K861060 + 0.1% oleic acid
| + | |
| - | <br>8: 5ml DH5α + BBa_K861060 + 1.0% oleic acid
| + | |
| - | <br>9: 5ml LB + 1% olive oil
| + | |
| - | <br>10: 5ml LB + 1% olive oil + chloramphenicol
| + | |
| - | <br>11: 5ml LB + 1% oleic acid
| + | |
| - | <br>12: 5ml LB + 1% oleic acid + chloramphenicol</p></h4>
| + | |
| - |
| + | |
| - | Samples were incubated at 37°c
| + | |
| - | | + | |
| - | <Br><Br>OD600 Readings were taken at hourly time points
| + | |
| - | <h4><p class="tab">100μl of each sample was pipetted into wells in a Transparent Greiner 96 microtitre plate.
| + | |
| - | <Br>As well as the 12 samples, 5 Control Wells (blanks) were created, without cells:
| + | |
| - | <br><Br>A: LB only
| + | |
| - | <br>B: 0.1% Oleic Acid only
| + | |
| - | <Br>C: 1.0% Oleic Acid only
| + | |
| - | <Br>D: 0.1% Olive Oil only
| + | |
| - | <Br>E: 1.0% Olive Oil only </p></h4>
| + | |
| - | | + | |
| - | How to use the Plate Reader
| + | |
| - | <h4><p class="tab">Turn on computer
| + | |
| - | <Br>Select ...
| + | |
| - | <Br>Select open plate icon
| + | |
| - | <br>Place microtitre plate into cassette holder
| + | |
| - | <br>Select close plate icon
| + | |
| - | <br>Open lid of plate reader
| + | |
| - | <br>Twist wheels - for OD select white and red wires, for fluorescence select yellow and blue wires
| + | |
| - | <br>Select spanner icon on program to choose between OD and fluorescence
| + | |
| - | <br>Select Measure icon
| + | |
| - | <br>Edit ….
| + | |
| - | <br>Select Excel icon
| + | |
| - | <br>Open A600 for OD, RFP Charlotte for Fluoresence
| + | |
| - | <br>Select update icon
| + | |
| - | <br>Scroll down to Table 2 to view results </p></h4>
| + | |
| - | | + | |
| - | <h5>Friday 11<sup>th</sup> July</h5>
| + | |
| - | | + | |
| - | <u>FadR assay analysis</u>
| + | |
| - | | + | |
| - | <h1> Week 4 </h1>
| + | |
| - | <h5>Monday 14<sup>th</sup> July </h5>
| + | |
| - |
| + | |
| - | Make <a href="#protocol3"></a>overnight</a> cultures.
| + | |
| - | <h4><p class="tab">
| + | |
| - | | + | |
| - | <li><a href="#biobrick1">DH5a + pSB3k3</a>
| + | |
| - | <li><a href="#biobrick1">DH5a + BBa_K861061</a>
| + | |
| - | <li><a href="#biobrick1">DH5a + BBa_K861060</a> x2
| + | |
| - | <li><a href="#biobrick1">DH5a</a>
| + | |
| - | <li><a href="#biobrick1">DH5a + pJeT 1.2 TliA</a>
| + | |
| - | <li><a href="#biobrick1">DH5a + pJeT 1.2 KerUS</a> </p></h4>
| + | |
| - | | + | |
| - | | + | |
| - | <h5>Tuesday 15<sup>th</sup> July </h5>
| + | |
| - |
| + | |
| - | Overnights of pJet 1.2 KerUS and TliA containing cells didn't grow, and so the time was used to make DNA stocks of BBa_K861060 and BBa_K861061.
| + | |
| - | | + | |
| - | <Br>Overnights were <a href="#protocol5">Miniprepped</a> and then <a href="#protocol8">Nanodropped</a>.
| + | |
| - | <br>dH<sub>2</sub>O was used instead of the elution buffer in the miniprep kit.
| + | |
| - | <h4><p class="tab">
| + | |
| - | <Br>FadR - 80.6ng/μl
| + | |
| - | <br>FadR - 75.9ng/μl
| + | |
| - | <br>RFP - 50.3ng/μl </p></h4>
| + | |
| - | | + | |
| - | Make <a href="#protocol3">Overnights</a> of pJet 1.2 + TliA and KerUS.
| + | |
| - | | + | |
| - | <h5>Wednesday 16<sup>th</sup> July </h5>
| + | |
| - |
| + | |
| - | <a href="#protocol5">Miniprep</a> Tlia and KerUS cells.
| + | |
| - | | + | |
| - | | + | |
| - | <Br><a href="#protocol8">Nanodrop</a>
| + | |
| - | <h4><p class="tab">
| + | |
| - | TliA - 522.0ng/μl
| + | |
| - | <bR>Kerus - 264.9ng/μl</P></H4>
| + | |
| - | | + | |
| - | <a href="#protocol13">Restriction Digests</A> were made with TliA, KerUS and FadR containing plasmids.
| + | |
| - | <Br>The following amounts of DNA were cut with restriction enzymes and the final reaction mixture bought up to 50μl.
| + | |
| - | <h4><p class="tab">
| + | |
| - | 2μl Tlia
| + | |
| - | <Br>4μl KerUS
| + | |
| - | <Br>12.5μl FadR
| + | |
| - | </p></h4>
| + | |
| - | Run a <a href="#protocol6">Gel</a> to seperate out the restriction fragments.
| + | |
| - | <br>Do a <a href="#protocol18">Gel extraction</a> on the bands which are the correct size. The DNA was eluted into dH<SUB>2</SUB>O.
| + | |
| - | <a href="#protocol13">Nanodrop</a>
| + | |
| - | <h4><p class="tab"> TliA - 5.7ng/μl
| + | |
| - | <Br>KerUS - 7.0ng/μl
| + | |
| - | <Br>FadR - 5.7ng/μl</p></h4>
| + | |
| - | | + | |
| - | A <a href="#protocol6">Ligation</a> reaction was set up containing either cut TliA or KerUS and FadR vector.
| + | |
| - | <Br> After an hour, the reaction mix was added to chemically competent DH5α which were <a href="#protocol6">transformed</a> and plated on antibiotic containing LB agar plates.
| + | |
| - | <br>Plates were incubated overnight at 37°C
| + | |
| - | | + | |
| - | | + | |
| - | <h5>Thursday 17<sup>th</sup> July </h5>
| + | |
| - | No positve colonies from the TliA and KerUS with FadR vector transformation
| + | |
| - | <Br><Br><a href="#protocol13">Day cultures</a> of BBa_861060, BBa_861061 and pSB3k3 were made.
| + | |
| - | <br>Once grown (leave for around 5 hours), 10μl of these cells were then plated onto either 25μl 1% Oleic Acid, 25μl 1% Olive Oil or 25μl LB agar plates.
| + | |
| - | <Br> Cells were left in the incubator overnight.
| + | |
| - | <h5>Thursday 17<sup>th</sup> July </h5>
| + | |
| - | No zone of clearing observed on olive oil containing plates
| + | |
| - | <Br><Br>
| + | |
| - | <h1>Week 5</h1>
| + | |
| - | | + | |
| - | <b> Tecan data </b>
| + | |
| - | A second run was done on the Tecan. Three different cells were used;
| + | |
| - | <h4><p class="tab"> BBa_k861060
| + | |
| - | <Br>BBa_k861061
| + | |
| - | <br>pSB3k3</p></h4>
| + | |
| - | | + | |
| - | Each of these cells were added to solutions containing 0.5% Olive oil, 1% Olive oil and 2% Olive oil. Solutions without cells added were also used as a control.
| + | |
| - | <Br> 200μl of each solution was pipetted into a 96 well plate with a clear bottom and white wells.
| + | |
| - | <Br>All unused wells were filled with LB to prevent evaporation of samples in the central wells.
| + | |
| - | <b> Fluorescence Microscopy </b>
| + | |
| - | <br><Br>Set up <a href="#protocol3">overnights</a> of the following;
| + | |
| - | <h4><p class="tab">
| + | |
| - | PSB3K3
| + | |
| - | <bR>pSB3K3 + 1% Olive Oil
| + | |
| - | <bR>pSB3K3 + 1% Oleic Acid
| + | |
| - | <br>BBa_K861060
| + | |
| - | <Br>BBa_K861060 + 1% Olive Oil
| + | |
| - | <br>BBa_K861060 + 1% Oleic Acid</p></h4>
| + | |
| - | | + | |
| - | | + | |
| - | <br> Add 10μl overnight cultures to microscope slide cover slips, allow to dry.
| + | |
| - | <Br> add 10μl 4% paraformaldehyde, dry for 30 minutes.
| + | |
| - | <Br>Wash 3 x with PBS buffer. Apply 10μl Prolong Gold Antifade Mountant with DAPI (Life Technologies). Fix to microscope slide, leave to dry overnight.
| + | |
| - | <br> Use fluorescence Microscope to visualise cells with DAPI and RFP suitable channels. Maintain the same exposure time between images so they are comparable.
| + | |
| - | | + | |
| - | | + | |
| - | <Br><Br>Following fresh ligation and transformation positive colonies were achieved for lipase with fadR responsive promoter in vector. Folllowing <a href="#protocol5">restriction digestion</a> with xbaI and pst1. Negative result.
| + | |
| - | <br><Br><a href="#protocol5">Overnights</a> of BBa_k861060, BBa_k861061 x2, TliA x2, PSB3K3 pellets frozen for minipreps later
| + | |
| - | | + | |
| - | <h1>Week 6</h1>
| + | |
| - | <h5>Monday 28<sup>th</sup> July</h5>
| + | |
| - | | + | |
| - | <a href="#protocol3">Miniprep</a> of overnights from Friday (BBa_k861060, BBa_k861061 x2, TliA x2, PSB3K3)
| + | |
| - | <h4><p class="tab">Plasmid DNA stored in -20</p></h4>
| + | |
| - | Produce 6 flasks of <a href="#protocol3">LB agar</a>.
| + | |
| - | <br><Br><a href="#protocol3">Overnights</a> of the following;
| + | |
| - | <h4><p class="tab">KerUS
| + | |
| - | <Br>TliA
| + | |
| - | <br><a href="#biobrick7">BBa_k823017</a>
| + | |
| - | <br>PSB3k3</p></h4>
| + | |
| - | | + | |
| - | | + | |
| - | <h5>Tuesday 29<sup>th</sup> July</h5>
| + | |
| - | | + | |
| - | Make up new stocks of <a href="#protocol20">soluble Olive oil (in Triton)</a>.
| + | |
| - | <br><Br>Make up 8 of agar plates with tributyrin in order to test lipase activity:
| + | |
| - | <h4><p class="tab">Differing concentrations of Tributyrin was added to either full strength or dilute LB agar. Any Olive oil was added to the empty plate and swirled to mix.
| + | |
| - | | + | |
| - | <Br><Br>
| + | |
| - | 0.5% tributyrin
| + | |
| - | <Br>0.5% tributyrin + 2% Olive Oil
| + | |
| - | <Br>1% tributyrin
| + | |
| - | <Br>1% tributyrin + 2% Olive Oil</p></h4>
| + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | <br><Br><b>Make M9 Media</b>
| + | |
| - | | + | |
| - | <br>Too 800ml distilled H<sub>2</sub>O add;
| + | |
| - | <h4><p class="tab"> 44g of Na2.2H2O
| + | |
| - | <br>15g of KH2.H2PO4
| + | |
| - | <Br>2.5g of NaCl
| + | |
| - | <Br>5g of NH4Cl</p></h4>
| + | |
| - | Pour 800ml of distilled H<sub>2</sub>O into 1L measuring cylinder
| + | |
| - | <Br>Disolve with magnetic flea and stirrer
| + | |
| - | <Br>Transfer media into 2 durans and autoclave.
| + | |
| - | | + | |
| - | <br><br>To 10mL water add either; <h4><p class="tab">1.2g of MgSO4
| + | |
| - | <Br> 1.1g of CaCl2</p></h4>
| + | |
| - | | + | |
| - | <Br>Shake/invert until salts dissolve
| + | |
| - | <Br>Transfer into conical flasks, cover with cotton wool and foil, autoclave.
| + | |
| - | | + | |
| - | <br><Br>Make <a href="#protocol7">Glycerol stocks</a> of pJet 1.2 KerUS, pJet 1.2 TliA, PSB3K3, BBa_K823017.
| + | |
| - | <br><br>Made 6 <a href="#protocol7">LB Agar</a> stocks.
| + | |
| - | <br><br><a href="#protocol7">Nanodrop</a> of pJet 1.2 TliA and pJet 1.2 Lipase, BBa_k861060
| + | |
| - | | + | |
| - | <br><a href="#protocol5">Restriction</a> and <a href="#protocol5">digest</a> of pJet 1.2 TliA and pJet 1.2 Lipase with BBa_k861060. <a href="#protocol5">Transform</a> into DH5a.
| + | |
| - | | + | |
| - | | + | |
| - | <h5>Wednesday 30<sup>th</sup> July </h5>
| + | |
| - | <br> One positive colony for TliA in the FadR vector in DH5a -ovenight culture of this
| + | |
| - | | + | |
| - | <h5>Thursday 31<sup>st</sup> July </h5>
| + | |
| - | | + | |
| - | <a href="#protocol5">Miniprep</a> the potential TliA in the FadR vector.
| + | |
| - | | + | |
| - | <br><a href="#protocol8">Nanodrop</a> 54ng/μl
| + | |
| - | | + | |
| - | <a href="#protocol8">Restriction digest</a> of potential TliA in the FadR vector with NdeI and EcoR1 to confirm - failed
| + | |
| - | | + | |
| - | <Br>Make <a href="#protocol8">glycerol stocks</a> of overnights potential TliA in the FadR vector
| + | |
| - | | + | |
| - | <h5>Friday 1<sup>st</sup> August </h5>
| + | |
| - | | + | |
| - | <a href="#protocol8">Restriction digest</a> undertaken.
| + | |
| - | <br>TliA and a construct thought to contain the gene for TliA in the FadR vector were <a href="#protocol5">restricted</a> using Xba1, Nde1 and both.
| + | |
| - | | + | |
| - | <a href="#protocol8">A gel</a> of the restriction digest was made
| + | |
| - | | + | |
| - | <br>The gel showed that the fadR plasmid did not contain the lipase.
| + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | <h1>Week 7</h1>
| + | |
| - | | + | |
| - | <br><b>ABC transporters into cells </b>
| + | |
| - | <a href="#protocol3">Overnights</a> of both ABC Transporters (BBa_K258008, BBa_K215107), T1SS signal-strep binding tag (BBa_K215002).
| + | |
| - | <br> <a href="#protocol5">Miniprep</a> the above plasmids and <a href="#protocol5">transform</a> into stocks of <a href="#protocol5">chemically competant</a> DH5a.
| + | |
| - | <br> Grow overnights of positive colonies, make competant and store at -80 in preperation for the transformation of a lipase containing plasmid.
| + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | <br><b>Cloning</b>
| + | |
| - | <br><a href="#protocol5">Miniprep</a> the pJet 1.2 TliA and promoters BBa_J23101, BBa_J23110 and BBa_J23100.
| + | |
| - | <Br>Elute into 50μ of dH<sub>2</sub>0.
| + | |
| - | | + | |
| - | <Br><a href="#protocol5">Nanodrop</a>
| + | |
| - | <h4><p class="tab">TliA - 103ng/μl
| + | |
| - | <Br>BBa_J23101 - 278ng/μl
| + | |
| - | <Br>BBa_J23110 - 408ng/μl
| + | |
| - | <br>BBa_J23100 - 380ng/μl</p></h4>
| + | |
| - | | + | |
| - | <Br>Carry out a <a href="#protocol5">Restriction digest</a> on the TliA and all three promoter plasmids - already had digested kerUS.
| + | |
| - |
| + | |
| - | <Br><Br>Slight variation to usual protocol: After 2 hours, 3μl CIP was added to the promoter plasmids and then returned to the heat block for another hour.
| + | |
| - | <Br>After 2 hours, the lipase was removed from the heat block.
| + | |
| - | | + | |
| - | <a href="#protocol5">Make a gel</a>
| + | |
| - | <Br>Restriction digestion mixes were all run on a DNA agarose gel and the correct sized bands cut.
| + | |
| - | | + | |
| - | <br>DNA was <a href="#protocol5">extracted</a> from the gel.
| + | |
| - | <br>The TliA and kerUS inserts were <a href="#protocol5">ligated</a> into each of the three different promoter strength harbouring vectors.
| + | |
| - | <Br>The ligation was left in the fridge overnight.
| + | |
| - | <br>Ligation mixes were <a href="#protocol5">transformed</a> into NEB5a cells
| + | |
| - | <br>Positive colonies were seen for TliA and kerUS in the BBa_J23110 containing vectors
| + | |
| - | <br>Colony PCR confirmed that TliA and kerUS had successfully been cloned into the BBa_J23110 vector
| + | |
| - | <br>These colonies were grown in liquid culture and <a href="#protocol5">-80°C stocks</a> were created
| + | |
| - | | + | |
| - | <br><Br>
| + | |
| - | <b>Tecan run with minimal media</b>
| + | |
| - | <br>DH5a plus the following plasmids were used for the tecan runs
| + | |
| - | <p class="tab">FadR-Rfp
| + | |
| - | <br>FadR
| + | |
| - | <br>J23110</p>
| + | |
| - | <br>2 x 50ml falcon tubes of M9 media was made
| + | |
| - | <p class="tab">35ml of dH<sub>2</sub>O was added from duran
| + | |
| - | <br>10ml of M9 salts was added from duran
| + | |
| - | <Br>2ml of casamino acids was added from falcon tube
| + | |
| - | <Br>100µl of MgSO<sub>4</sub> was added from 50ml conical flask
| + | |
| - | <Br>1ml of 20% glucose solution was added to one falcon tube, 1ml of dH<sub>2</sub>O was added to the
| + | |
| - | other
| + | |
| - | <br>5µl of CaCl<sub>2</sub> was added from 50ml conical flask
| + | |
| - | <br>topped up with dH<sub>2</sub>O to make 50ml solution</p></h4>
| + | |
| - | | + | |
| - | Lipid was added to the M9 Solution
| + | |
| - | <br>Total volume created = 4ml, in 6ml bijou tubes
| + | |
| - | <br>Since the Olive oil:Triton X-100 ratio was 1:2, the following concentrations were made:
| + | |
| - | <br><br><table style="width:50%; color:#696969">
| + | |
| - | <tr>
| + | |
| - | <th>Solution</th>
| + | |
| - | <th>1%</th>
| + | |
| - | <th>2%</th>
| + | |
| - | <th>3%</th>
| + | |
| - | <th>4%</th>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td>M9 Media</td>
| + | |
| - | <td>4.0ml</td>
| + | |
| - | <td>3.88ml</td>
| + | |
| - | <td>3.76ml</td>
| + | |
| - | <td>3.52ml</td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td>Olive oil:Triton</td>
| + | |
| - | <td>0μl</td>
| + | |
| - | <Td>120μl</td>
| + | |
| - | <td>240μl</td>
| + | |
| - | <td>480μl</td>
| + | |
| - | </tr>
| + | |
| - | </table>
| + | |
| - | | + | |
| - | The 4.0% stock was made from the falcon tube without glucose added as a supplementary carbon source.
| + | |
| - | <br> the 0.0%, 1.0% and 2.0% were made from the falcon tube with glucose added as a supplementary carbon source
| + | |
| - | | + | |
| - | <Br><Br>Transformed cells & antibiotics were added to all 4 concentrations of M9/Lipid media.
| + | |
| - | <Br><Br>96-Well plate was made.
| + | |
| - | <br>200μl of each solution containing the 3 different promoters were pipetted into 4 wells to create 4 technical repeats.
| + | |
| - | <Br>200μl of each solution devoid of cells were pipetted into 3 wells in the region of the plate (B-G, 10-11)
| + | |
| - | <Br><Br>
| + | |
| - | | + | |
| - | <h1>Week 9</h1>
| + | |
| - | <h5>Monday 18<sup>th</sup> August</h5>
| + | |
| - | | + | |
| - | Make <a href="#protocol3">overnights</a> of <a href="#biobrick1">kerUS</a> and <a href="#biobrick1">BBa_J23110</a>
| + | |
| - | | + | |
| - | <h5>Tuesday 19<sup>th</sup> August </h5>
| + | |
| - | | + | |
| - | Make 4 <a href="#protocol15">day cultures</a>;
| + | |
| - | <h4><p class="tab"> 5ml KerUS + Nail
| + | |
| - | <br> 5ml KerUS + Hair
| + | |
| - | <br> 5ml BBa_J23110 + Nail
| + | |
| - | <br> 5ml BBa_J23110 + Hair</p></h4>
| + | |
| - | These were left in the shaking incubator for the cells to grow and produce and secrete keratinase.
| + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | <br><br>Autoclave hair, nail and feather samples to sterilise. This was done in glass beakers with foil 'lids'.
| + | |
| - | | + | |
| - | <br><br><a href="#protocol23">Restreak</a> KerUS and BBa_J23110.
| + | |
| - | | + | |
| - | <br><Br>Make <a href="#protocol3">overnights</a> of kerUS and BBa_J23110.
| + | |
| - | | + | |
| - | <h5>Wednesday 20<sup>th</sup> August</h5>
| + | |
| - | <a href="#protocol3">Overnights</a> of <a href="#biobrick1">BL21</a>.
| + | |
| - | | + | |
| - | | + | |
| - | <h5>Thursday 21<sup>st</sup> August</h5>
| + | |
| - | Make 2 5ml <a href="#protocol15">day cultures</a> of BL21.
| + | |
| - | <br> Allow around 3 hours for them to reach the exponential growth phase.
| + | |
| - | <br><a href="#protocol16">Take an OD reading </a>
| + | |
| - | <br>If the OD600 is over 0.6, use these innoculations to produce <a href="#protocol4">Chemically competant</a> cells.
| + | |
| - | | + | |
| - | <br><Br><a href="#protocol5">Miniprep</a> KerUS BBa_J23110
| + | |
| - | <br><br><a href="#protocol11">Transform</a> the chemically competant BL21 strain using 250ng of the purified KerUS BBa_J23110 DNA.
| + | |
| - | <br><Br>Plate the following;
| + | |
| - | <h4><p class="tab"> 100μl of control onto an antibiotic free plate.
| + | |
| - | <Br>100μl of control onto an ampicillin plate.
| + | |
| - | <Br>100μl of transformant onto an ampicillin plate.
| + | |
| - | <Br>Remaining 900μl spun down and resuspended in 100μl of LB and plated onto an ampicillin plate.
| + | |
| - | | + | |
| - | </p></h4>
| + | |
| - | <br><a href="#protocol5">Transform</a> TliA BBa_J23110 into stocks of ABC transporter (BBa_K258008) containing competent cells
| + | |
| - | <br><a href="#protocol11">Pour plates</a> containing Lb Agar, Lb Agar containing 1% casein, Lb Agar containing 1% gelatin and Agar containing 1% gelatin.
| + | |
| - | | + | |
| - | <h5>Friday 22<sup>nd</sup> August</h5>
| + | |
| - | <br>Positive colonies of all transformations
| + | |
| - | <br><a href="#protocol3">Overnights</a> of positive colonies of cells containing <a href="#biobrick1">TliA/BBa_J23110/BBa_K258008</a>, KerUs/BBa_J23110 and BBa_J23110
| + | |
| - |
| + | |
| - | <h5>Saturday 23<sup>nd</sup> August</h5>
| + | |
| - | | + | |
| - | | + | |
| - | <br>Plate out KerUs containing overnights onto Casein, Gelatin and Gelatin LB agar plates. BBa_J23110 was used as a negative control. <i>Staphylococcus aureus</i> was used as a positive control.
| + | |
| - | <br> Incubate for 8 hours, then flood plates in saturated ammonium sulphate.
| + | |
| - | <br><Br>
| + | |
| - | | + | |
| - | <h1>Week 10</h1>
| + | |
| - | <h5>Monday 25<sup>th</sup> August</h5>
| + | |
| - | Make <a href="#protocol3">overnights</a> of KerUS/BBa_J23110, tliA/BBa_J23110, tliA/BBa_J23110/BBa_K258008 and BBa_J23110.
| + | |
| - | <Br>Autoclave four 1 Litre conical flasks each containing 100ml of LB Broth.
| + | |
| - | | + | |
| - | <h5>Tuesday 26<sup>th</sup> August</h5>
| + | |
| - | Innoculate each of the autoclaved flasks with 1ml of each of the overnights. Add the correct antibiotic to each.
| + | |
| - | <br>Incubate at 30°C to promote protein expression.
| + | |
| - | <Br>Give 5 or 6 hours for the cells to reach exponential growth phase and produce an adequate amount of protein to assay.
| + | |
| - | | + | |
| - | <Br><Br>After 6 hours, samples were taken for analysis by protein gel.
| + | |
| - | <h4><p class="tab"> Into 4 eppendorfs, pipette 1.5 ml of each innoculation.
| + | |
| - | <br>These are spun down and the supernatant removed into a second tube. Following the addition of 150μl trichloroacetic acid (TCA) and kept in the 4 degree overnight.
| + | |
| - | <br><br> Into 4 more eppendorfs, pipette 0.5ml of each innoculation.
| + | |
| - | <br>These are spun down, the supernatant poured away, the pellet labelled and kept in the -20 degree.
| + | |
| - | <Br><Br>Remaining culture was centrifuged, sterile filtered and loaded into Vivaspin 20 10,000 MWCO columns (GE Life Science) (tliA without exporter was excluded) add 5ml of culture supernatant. These were spun down at 3,500 rpm for 8 minutes.
| + | |
| - | <br>Supernatant that had passed through the column was discarded, and more supernatant was added into the top of the column. This was repeated until around 15ml of supernatant had passed through the column.
| + | |
| - | <Br>Around 1ml of concentrated supernatant was then removed from the top of the column.
| + | |
| - | | + | |
| - | <br><br> <a href="#protocol5">Overnight</a> liquid cultures of cells containing kerUS (no promoter), tliA/BBa_J23110 and pSB1C3
| + | |
| - | | + | |
| - | <h5>Wednesday 27<sup>th</sup> August</h5>
| + | |
| - | To prepare the protein sample to be loaded onto a gel
| + | |
| - | <Br><h4><p class="tab">To the cell pellet add 100μl 2X SDS buffer. Resuspend the cells in this solution.
| + | |
| - | <Br>Centrifuge the 1.5ml of supernatant/TCA mix for 15 minutes at 13,300rpm, remove supernatant, resuspend pellet in 900μl acetone, repeat centrifugation step, remove supernatant, allow remaining acetone to evaporate then add 50μl of SDS buffer (a ratio of ~1:20)
| + | |
| - | <br>To the 1ml concentrated supernatant, add 1ml of SDS buffer (a ratio of 1:1)
| + | |
| - | <br>Place all of these samples into a heat block at 95°C for 5 minutes. The cell pellet may need to be vortexed and left for an additional 5 minutes.</p></h4>
| + | |
| - | | + | |
| - | <br><Br><a href="#protocol5">Make a 12% SDS-PAGE gel</a>. Load 5μl cell lysate samples and 15μl supernatant (concentrated or non-concentrated) onto the gel
| + | |
| - | <br>Stain the SDS-PAGE gel with InstantBlue (Expedeon) overnight
| + | |
| - | <br><br> <a href="#protocol5">Miniprep</a> plasmids from overnight cultures, set up <a href="#protocol5">restriction digests</a> with EcoRI and PstI (add CIP to the acceptor vector). Run donor mixes on a DNA gel and isolate DNA from bands of correct size
| + | |
| - | <br><a href="#protocol5">Ligate</a> isolated cut kerUS and tliA/J23110 with cut pSB1C3 overnight
| + | |
| - | | + | |
| - | <br><br>
| + | |
| - | <h5>Thursday 28<sup>th</sup> August</h5>
| + | |
| - | <br> <a href="#protocol5">Transform</a> both ligation mixes into NEB5a and <a href="#protocol5">plate</a> on chloramphenicol containing LB agar plates
| + | |
| - | <h5>Friday 29<sup>th</sup> August </h5>
| + | |
| - | <br>Positive colonies on both LB agar plates. <a href="#protocol5">Colony PCR</a> to confirm insertion of kerUS or tliA/J23110.
| + | |
| - | <br>Overnight liquid cultures of a positive colony from each.
| + | |
| - | | + | |
| - | | + | |
| - | <h5>Saturday 30<sup>th</sup> August</h5>
| + | |
| - | <a href="#protocol5">Miniprep</a> overnight cultures. Send some for sequencing with sequencing primers. Once confirmed send on to the BioBricks Registry </div>
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Run a <a href="#protocol6">DNA gel</a> using the purified DNA collected yesterday to check DNA is pure.
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