Difference between revisions of "Team:XJTLU-CHINA/Protocols"

 
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<div class="container">
 
     <h1>Basic Molecular Biology Techniques:</h1>
 
     <h1>Basic Molecular Biology Techniques:</h1>
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     <h3>PCR using Q5 Hi-Fi DNA polymerase Master Mix</h3>
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     <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_PCR"><h3>PCR using Q5 Hi-Fi DNA polymerase Master Mix</h3></a>
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    <p>Primer 1:  5’-GCACTCACCATGGGTACTGCAGAATTCGCGGCCGCTTCT-3’</p>
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     <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_PCR_purification"><h3>PCR purification using QIAquick PCR purification kit</h3></a>
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     <p>Primer 2: 5’-TAGTGGTACCGCATGCCTGCACTGCAGCGGCCGCTACT-3’</p>
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     <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Gel_extraction"><h3>Gel Extraction</h3></a>
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    <p>Reaction setup:</p>
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     <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Gibson_Assembly"><h3>Gibson assembly</h3></a>
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     <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2017/6/6f/Fluorescein_standard_curve.png">
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     <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Digestion"><h3>Digestion (NEB enzymes)</h3></a>
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    <p>The template DNA should keep an amount of 1-1000 ng</p>
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     <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Ligation"><h3>Ligation</h3></a>
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    <p>Thermocycler conditions</p>
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     <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Transformation"><h3>Transformation of plasmids in <i>E.coli</i> DH5ɑ</h3></a>
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     <p>Clarification:</p>
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     <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Isolation"><h3>Isolation of plasmids in <i>E.coli</i> DH5ɑ</h3></a>
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    <ol>
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     <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Preparation_Competent"><h3>Preparation of competent <i>Lactococcus lactis</i></h3></a>
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    <li>The sequences in red are where the primers anneal with the biobrick affixes. And the 5’overhangs side with the sequences in red are designed for Gibson Assembly with linearized pNZ8148 digested by PstI due to a 21-nucleotide overlap between them.</li>
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     <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Electroporation"><h3>Electroporation for <i>Lactococcus lactis</i></h3></a>
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     <li>The reason of setting two different cycles is that, at the beginning of PCR, the annealed parts between primers and templates are only sequences in red. As PCR proceeds, 5’overhangs are added to the entire genes, so the rest reaction of PCR becomes the one between the whole primers and the whole genes, because of which the Tm varies compared to the former.</li>
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     <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Plasmid_DNA_isolation"><h3>Plasmid DNA isolation from <i>Lactococcus lactis</i></h3></a>
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    </ol>
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      <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Dot_blot"><h3>Dot Blot</h3></a>
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     <p>Applications in this project: Amplification of BBa_K2309028, BBa_K2309003 and BBa_K2309004</p>
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<hr>
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    <h3>PCR purification using QIAquick PCR purification kit</h3>
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    <ol>
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    <li>Add 500 μl Qiagen buffer PB</li>
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     <li>Spin through a column twice, discard flow-through</li>
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    <li>Wash 1x with 700 μl buffer PB</li>
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    <li>Wash 2x with 760 μl buffer PE</li>
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     <li>Discard liquid, spin dry at 17000g for 3 min</li>
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    <li>Elute into a new tube twice with 50 μl of TE (100 μl total)</li>
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    </ol>
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    <p>(Can be also referred to from iGEM protocol)</p>
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     <h3>Gel extraction</h3>
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    <h3>Gibson Assembly</h3>
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    <h3>Digestion (NEB enzymes)</h3>
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     <h3>Ligation</h3>
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    <h3>Transformation of plasmids in E.coli DH5ɑ</h3>
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    <h3>Isolation of plasmid in E.coli DH5ɑ</h3>
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     <h3>Preparation of competent Lactococcus lactis</h3>
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    <h3>Electroporation for L. lactis</h3>
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    <h3>Plasmid DNA isolation from Lactococcus lactis</h3>
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     <h1>Anti-microbial peptides assay:</h1>
 
     <h1>Anti-microbial peptides assay:</h1>
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     <h1>Nisin induction of gene expression in Lactococcus lactis:</h1>
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     <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_CFU_measurement"><h3>Pre-experiment: CFU measurement</h3></a>
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     <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Inhibition_ring"><h3>Inhibition disc</h3></a>
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    <ol>
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    <li>Add 100μl of LUDOX into wells A1, B1, C1, D1.</li>
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    <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_AMPs_efficiency_MIC_assay"><h3>AMPs minimal bactericidal concentration (MBC) assay</h3></a>
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    <li>Add 100μl of H<sub>2</sub>O into wells A2, B2, C2 D2.</li>
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    <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Biofilm_formation"><h3>Biofilm formation test</h3></a>
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    <li>Measure absorbance 600 nm of all samples in a microplate reader and record the data.</li>
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    <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Growth_curve"><h3>Growth curve measurement</h3></a>
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    <li>Add 1ml of LUDOX into cuvette for the measurement of OD<sub>600</sub> in spectrophotometer.</li>
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    </ol>
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    <h3>Fluorescein fluorescence standard curve</h3>
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    <ol>
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    <li>Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.</li>
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     <li>Prepare 2x fluorescein stock solution (100μM) by re-suspending fluorescein in 1ml pf 1x PBS.</li>
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    <li>Dilute the 2x fluorescein stock solution with 1x PBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50μM.</li>
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    <li>Add 200μl of 1x fluorescein stock solution into well A1, B1, C1 D1 of 96-well plate and 100μl of PBS to A2, B2, C2, D2…A12, B12, C12, D12.</li>
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    <li>Transfer 100μl of solution from A1 into A2 and mix by pipetting up and down three times, and then continue the same procedure and transfer from A2 to A3, from A3 to A4……Finally, transfer 100μl from A11 into liquid waste. Repeat dilution series for rows B, C, D.</li>
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    <li>Measure fluorescence of all samples in a microplate reader and record the data.</li>
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    </ol>
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    <h3>Cell measurement</h3>
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    <ol>
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    <li>Day 1: Transform <i>Escherichia coli</i> DH5α with these following plasmids:
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        <ul>
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        <li>Positive control </li>
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        <li>Negative control </li>
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        <li>Test Device 1: J23101+I13504</li>
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        <li>Test Device 2: J23106+I13504 </li>
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        <li>Test Device 3: J23117+I13504 </li>
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        <li>Test Device 4: J23101.BCD2.E0040.B0015</li>
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        <li>Test Device 5: J23106.BCD2.E0040.B0015</li>
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        <li>Test Device 6: J23117.BCD2.E0040.B0015</li>
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        </ul>
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    </li>
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    <li>Day 2: Pick 2 colonies from each of plate and inoculate it on 5ml LB with 25μg/ml chloramphenicol for about 17 hours (3:00pm to 10am, next day), at 37℃ and 220rpm.</li>
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    <li>Day 3: Cell growth, sampling, and assay
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        <ol type="a">
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        <li>Take 200μl of each overnight culture mixed with 800μl water to make a 5-fold dilution.</li>
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        <li>Measure OD<sub>600</sub> of the overnight cultures and record the data.</li>
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        <li>Dilute the cultures to a target OD<sub>600</sub> of 0.02 in 12ml LB medium + Chloramphenicol in 50ml falcon tube. </li>
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        <li>Incubate the cultures at 37°C and 220rpm. </li>
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        <li>Take 500μl samples of the cultures at 0, 2, 4, and 6 hours of incubation.</li>
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        <li>Place samples on ice.</li>
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        <li>Take 100μl of each sample into 96-well plate to get values of fluorescence and OD<sub>600</sub>. For each sample, there were 4 replicates.</li>
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        </ol>    
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    </li>
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    </ol>
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    <p>We laid out sample as shown in following picture:</p>
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    <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2017/a/a7/Sample.png">
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     <hr>
 
     <hr>
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     <h1>Result</h1>
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     <h1>Nisin induction of gene expression in <i>Lactococcus lactis</i>:</h1>
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    <h3>The OD<sub>600</sub> reference</h3>
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     <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Testing_sensing_pathway"><h3>Testing of <i>S. aureus</i> sensing pathway</h3></a>
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    <div class="row">
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     <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Autolysis_test"><h3>Autolysis test</h3></a>
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        <div class="col-sm-6">
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</div><!-- container --!>
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        <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2017/2/2a/The_od600_reference.png">
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        </div>
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        <div class="col-sm-6">
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        <p>The average Abs<sub>600</sub> of LUDOX and H<sub>2</sub>O were calculated and showed in the table. The corrected Abs<sub>600</sub> is obtained by subtracting the Abs<sub>600</sub> of H<sub>2</sub>O from the Abs<sub>600</sub> of LUDOX. The reference OD<sub>600</sub> was measured from 1ml LUDOX using a reference spectrophotometer at 600nm. The value of OD<sub>600</sub>/Abs<sub>600</sub> is 4.25.</p>
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        </div>
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     </div>
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    <h3>Fluorescein fluorescence standard curve</h3>
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    <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2017/6/6f/Fluorescein_standard_curve.png">
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    <p class="form"><b>Fig 1.</b>&nbsp;&nbsp;&nbsp;&nbsp;The fluorescence standard curve of fluorescein
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    </br>The measured fluorescence was plotted against the concentration of fluorescein.</p>  
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    <p>The fluorescence signal increased 150 A.U. with the concentration of fluorescein increased 1μM at low concentration, from 0 to 20μM, and the slope of the curve was gradually decreasing as the concentration increased.</p>
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    <h3>Cell measurement</h3>
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     <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2017/d/d1/Od600_of_different_devices_over_time.png">
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    <p class="form"><b>Fig 2.</b>&nbsp;&nbsp;&nbsp;&nbsp;OD<sub>600</sub> against the incubation time
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    </br>This figure showed the OD<sub>600</sub> of 16 samples with different devices during the incubation.</p>
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    <p>The growth rate of DH5α transformed with device 1 was significantly less than that of DH5α transformed with other devices, while this might have been an effect of the gene expression of device 1, but this can also happen due to human error.</p>
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    <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2017/8/81/Fluorescene_signals_from_different_devices_over_time.png">
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    <p class="form"><b>Fig 3.</b>&nbsp;&nbsp;&nbsp;&nbsp; Fluorescence signals from different devices over time
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    </br>In this figure, we give a curve of fluorescence signal of each sample at specific points (0, 2, 4 and 6 hours).</p>
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    <p>The culture of DH5α transformed with device 2 produced the highest fluorescent signal and followed by device 4 less than the positive control.</p>
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    <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2017/6/63/Fluorescein_productivity_of_different_devices.png">
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    <p class="form"><b>Fig 4.</b>&nbsp;&nbsp;&nbsp;&nbsp; GFP productivity of different devices</p>
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    <p>The blue, orange, grey and yellow bars correspondingly represented the ratio of GFP concerntration to OD<sub>600</sub> value at 0, 2, 4, 6 hours. The concertation of GFP was converted from fluorescence signal (in Figure 3), with calibration curve in Figure 2.</p>
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    <hr>
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    <h1>Discussion</h1>
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    <p>We observed that, among the 6 combinations of these 3 promoters (J23101, J23106 and J23117) and 2 RBS (B0034 and BCD2) in various devices, devices 2 and 4 produced high fluorescence signals in the bacterial cell population and device 1 had the strongest ability to produce GFP per cell which was companied with low growth. Based on the plot of [GFP]/OD<sub>600</sub>, either with RBSs B0034 or BCD2, the rank of the promoter efficiency (from strong to weak) is J23101, J23106 and J23117. When the promoters are the same, the strength of BCD2 is weaker than that of B0034. It was quite typical that, although the population of bacterial cells with device 1 had relatively low GFP level, the [GFP]/OD<sub>600</sub> of device 1 was the highest (except 0h) among all the samples. However, when merely considering the total quantity of GFP in bacterial population, obviously device 1 is not a good choice to produce GFP. That may be due to the overexpression of GFP in comsuming too many resources, leading to the lack of materials for cell growth.</p>
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    </div><!-- container --!>
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Latest revision as of 10:46, 26 November 2017

Protocols

Protocols

Basic Molecular Biology Techniques:

PCR using Q5 Hi-Fi DNA polymerase Master Mix

PCR purification using QIAquick PCR purification kit

Gel Extraction

Gibson assembly

Digestion (NEB enzymes)

Ligation

Transformation of plasmids in E.coli DH5ɑ

Isolation of plasmids in E.coli DH5ɑ

Preparation of competent Lactococcus lactis

Electroporation for Lactococcus lactis

Plasmid DNA isolation from Lactococcus lactis

Dot Blot

Anti-microbial peptides assay:

Pre-experiment: CFU measurement

Inhibition disc

AMPs minimal bactericidal concentration (MBC) assay

Biofilm formation test

Growth curve measurement

Nisin induction of gene expression in Lactococcus lactis:

Testing of S. aureus sensing pathway

Autolysis test

Collaborators and Supporters

Location

Rm 363, Science Building
Xi'an Jiaotong-Liverpool University
111 Ren'ai Road, Suzhou, China
215123

Get in touch

emali

igem@xjtlu.edu.cn

XJTLU-CHINA iGEM 2017