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Commit 2875bc26 authored by Yanhao Chen's avatar Yanhao Chen
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<!-- Ch and quantification-->
<div class="myPage-paragraph-headline-h3 expstep_name">Characterization and quantification of BC
production in <i>E. coli</i> EcNP
</div>
<div class="panel">
<div class="panel-heading" role="tab" id="heading68">
<h4 class="panel-title">
<a role="button" class="collapsed" data-toggle="collapse" href="#collapse68"
aria-expanded="true" aria-controls="collapse68">
Requirements
</a>
</h4>
</div>
<div id="collapse68" class="panel-collapse collapse" role="tabpanel">
<div class="panel-body">
<p>● Recombinant <i>E. coli</i> Nissle 1917 (EcNP) strain<br>
● LB liquid medium<br>
● Ampicillin (100 mg/mL)<br>
● Kanamycin (50 mg/mL)<br>
● 500 g/L glucose solution<br>
● 37 °C shaking incubator<br>
● 500 g/L Congo red solution<br>
● Dynamic fermentation BC standard<br>
● Tecan Infinite M200 microplate reader<br>
● 96-well plate<br>
</p>
</div>
</div>
</div>
<div class="myPage-paragraph-headline-h4 th4">
A. Determination of BC standard curve
</div>
<div class="panel">
<div class="panel-heading" role="tab" id="heading69">
<h4 class="panel-title">
<a class="collapsed" role="button" data-toggle="collapse" href="#collapse69"
aria-expanded="false" aria-controls="collapse69">
Protocol
</a>
</h4>
</div>
<div id="collapse69" class="panel-collapse collapse" role="tabpanel">
<div class="panel-body">
<p>
1. Prepare BC standard samples with concentrations of 0, 20, 40, 60, 80, and 100 mg/L.
Perform five parallel experiments for each concentration.<br>
2. Add 1 mL of BC standard solution to a 1.5-mL centrifuge tube, then add 1 μL of 500 g/L
Congo red solution. Immediately vortex the mixture for 10 s.<br>
<i>Note: Please mix well before using Congo red solution.</i><br>
3. Culture at room temperature for 30 minutes away from light.<br>
4. Centrifuge at 9000 <i>r.p.m.</i> for 7 minutes.<br>
5. Take 200 μL of the supernatant and transfer it to a 96-well plate. Measure OD490
values.<br>
6. Plot a standard curve with the absorbance values to BC concentrations.<br>
</p>
</div>
</div>
</div>
<div class="myPage-paragraph-headline-h4 th4">
B. Measurement of BC samples
</div>
<div class="panel">
<div class="panel-heading" role="tab" id="heading70">
<h4 class="panel-title">
<a class="collapsed" role="button" data-toggle="collapse" href="#collapse70"
aria-expanded="false" aria-controls="collapse70">
Before the experiment
</a>
</h4>
</div>
<div id="collapse70" class="panel-collapse collapse" role="tabpanel">
<div class="panel-body">
<p>
1. Inoculate the recombinant EcNP strains in 2% ratio into 10 mL of LB medium containing<br>
corresponding antibiotics and glucose (20 g/L) at 37 °C and 200 <i>r.p.m.</i>.<br>
2. Add IPTG at final concentrations of 0.5 mM to it when the OD600 reaches 0.8.<br>
3. Shake the cultures at 37 °C with shaking at 200 <i>r.p.m.</i> for several hours.<br>
<i>Note: Perform steps 1-3 for the negative control as well.</i>
</p>
</div>
</div>
</div>
<div class="panel">
<div class="panel-heading" role="tab" id="heading71">
<h4 class="panel-title">
<a class="collapsed" role="button" data-toggle="collapse" href="#collapse71"
aria-expanded="false" aria-controls="collapse71">
Protocol
</a>
</h4>
</div>
<div id="collapse71" class="panel-collapse collapse" role="tabpanel">
<div class="panel-body">
<p>
1. Combine 1 mL of bacterial culture with 1 μL of 500 g/L Congo Red solution in a 1.5-mL
centrifuge tube. Immediately vortex the mixture for 10 s.<br>
<i>Note: Please mix well before using Congo red solution.</i><br>
2. Culture at room temperature for 30 minutes away from light.<br>
3. Centrifuge at 9000 <i>r.p.m.</i> for 7 minutes.<br>
4. Take 200 μL of the supernatant and transfer it to a 96-well plate. Measure
OD<sub>490</sub> values to calculate concentration of BC.<br>
<i>Note: Be careful to ensure that the final data falls within the standard curve range.</i><br>
</p>
</div>
</div>
</div>
<div class="myPage-paragraph-headline-h4 th4">
C. Test of the optimal IPTG induction concentration
</div>
<div class="panel">
<div class="panel-heading" role="tab" id="heading72">
<h4 class="panel-title">
<a class="collapsed" role="button" data-toggle="collapse" href="#collapse72"
aria-expanded="false" aria-controls="collapse72">
Protocol
</a>
</h4>
</div>
<div id="collapse72" class="panel-collapse collapse" role="tabpanel">
<div class="panel-body">
<p>
1. Inoculate the recombinant EcNP strains in 2% ratio into 10 mL of LB medium containing
corresponding antibiotics and glucose (20 g/L) at 37 °C and 200 <i>r.p.m.</i>.<br>
2. Add IPTG at final concentrations of 0, 0.1, 0.5, and 1 mM to each system when the OD<sub>600</sub>
reaches 0.8.<br>
3. Take samples after 4 hours and compare the BC content in the bacterial culture.<br>
</p>
</div>
</div>
</div>
<div class="myPage-paragraph-headline-h3 expstep_name">Production and test of cross-linked
water-retention material
......
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