96-well plate, 1.5 ml EP tube, Black bottom 96-well plate, Centrifuge, Centrifuge tube
</p>
<p>
Procedure:
</p>
<p><b>Signal Sender</b></p>
...
...
@@ -1934,7 +1953,8 @@
</table>
<ul>
<li>19. Measure and record OD<sub>600</sub> as the value of 0 hours. Label and put them into the thermostatic shaker to culture. Start timing at the same time.</li>
<li>19. Measure and record OD<sub>600</sub> as the value of 0 hours. Label and put them into the thermostatic
shaker to culture. Start timing at the same time.</li>
<li> 20. Measure and record OD<sub>600 every 3 hours until culturing them for 48 hours.</li>
</ul>
...
...
@@ -1942,21 +1962,33 @@
<divclass="h2">Measurement of Growth Curve of the Three </div>
<li>Add 20 g of sucrose into the CoBG11 liquid medium to obtain CoBG11-sucrose liquid medium. Sterilize by the filter.</li>
<li>Seed initial cultures at a ratio of 1:100 for <i>E. coli</i> and <i>A. caulinodans</i>, and at a ratio of 1:20 for <i>S. elongatus</i> into 100 ml CoBG11-sucrose liquid medium in a thermostatic shaker(for <i>E. coli</i>, at 37℃, 220 rpm; for <i>A. caulinodans</i>, at 37℃, 180 rpm; for <i>S. elongatus</i>, at 30℃, 130 rpm with lamp tube).</li>
<li>Measure and record the OD value of each culture medium every 4h (<i>E. coli</i>), 6h(<i>A. caulinodans</i>), or 12h (<i>S. elongatus</i>).</li>
<li>Add 20 g of sucrose into the CoBG11 liquid medium to obtain CoBG11-sucrose liquid medium. Sterilize by the
filter.</li>
<li>Seed initial cultures at a ratio of 1:100 for <i>E. coli</i> and <i>A. caulinodans</i>, and at a ratio of
1:20 for <i>S. elongatus</i> into 100 ml CoBG11-sucrose liquid medium in a thermostatic shaker(for <i>E.
coli</i>, at 37℃, 220 rpm; for <i>A. caulinodans</i>, at 37℃, 180 rpm; for <i>S. elongatus</i>, at 30℃, 130
rpm with lamp tube).</li>
<li>Measure and record the OD value of each culture medium every 4h (<i>E. coli</i>), 6h(<i>A. caulinodans</i>),
or 12h (<i>S. elongatus</i>).</li>
</ol>
<div>
<divclass="h2">Immobilization of <i>E. coli</i> and <i>S. elongatus</i></div>
</div>
<p><b>Reagent</b>: Initial <i>E .coli</i> bacterial fluid, Initial <i>S .elongatus</i> bacterial fluid, Sodium alginate powder, Calcium chloride, CoBG11-sucrose liquid medium and CoBG11-acetate liquid medium(see 'Experiments on <i>A. caulinodans</i>'-'Measurement of Growth Curves'), Physiological saline, Milli-Q water</p>
<divclass="h3">Preparation of Sodium Alginate Solution</div>
...
...
@@ -1976,7 +2008,8 @@
<p>Procedure:</p>
<ol>
<li>Add 2.22 g calcium chloride to 100 ml Milli-Q water. Make the concentration of the calcium chloride reach 0.2 M.</li>
<li>Add 2.22 g calcium chloride to 100 ml Milli-Q water. Make the concentration of the calcium chloride reach
0.2 M.</li>
<li>Use the filter to filter the solution.</li>
</ol>
...
...
@@ -1987,10 +2020,17 @@
<p>Procedure:</p>
<ol>
<li>Centrifuge the microorganism fluid at 4000 rpm for 15 min. Remove the supernatant and weigh the wet weight.</li>
<li>For every 1 g cell, resuspend in 1 ml physiological saline, add 20 ml sodium alginate solution and stir until well combined.</li>
<li>Transfer the alginate-cell mixture into a small syringe (needle removed) and drips naturally into 20 ml 0.2 M calcium chloride solution. Meanwhile, stir with an electromagnetic stirrer.</li>
<li>Soak for 5 hours, then transfer the immobilized cells into CoBG11-sucrose medium (for <i>E. coli</i> and <i>S. elongatus</i>) or CoBG11-acetate medium (for <i>A. caulinodans</i>), culture in a shaker (for <i>E. coli</i>, at 37℃, 220 rpm; for <i>A. caulinodans</i>, at 37℃, 180 rpm; for <i>S. elongatus</i>, at 30℃, 130 rpm).</li>
<li>Centrifuge the microorganism fluid at 4000 rpm for 15 min. Remove the supernatant and weigh the wet weight.
</li>
<li>For every 1 g cell, resuspend in 1 ml physiological saline, add 20 ml sodium alginate solution and stir
until well combined.</li>
<li>Transfer the alginate-cell mixture into a small syringe (needle removed) and drips naturally into 20 ml 0.2
M calcium chloride solution. Meanwhile, stir with an electromagnetic stirrer.</li>
<li>Soak for 5 hours, then transfer the immobilized cells into CoBG11-sucrose medium (for <i>E. coli</i> and
<i>S. elongatus</i>) or CoBG11-acetate medium (for <i>A. caulinodans</i>), culture in a shaker (for <i>E.
coli</i>, at 37℃, 220 rpm; for <i>A. caulinodans</i>, at 37℃, 180 rpm; for <i>S. elongatus</i>, at 30℃, 130
rpm).
</li>
</ol>
<div>
...
...
@@ -2003,7 +2043,8 @@
<li>Transfer the immobilized cells into 1.5 ml EP tubes and weigh wet weight.</li>
<li>For every 1 g immobilized cell, add 1 ml 0.2 M phosphate buffer (pH=7.5).</li>
<li>Shake violently for 15 min until all solids dissolve.</li>
<li>Measure the OD<sub>600</sub> (for <i>E. coli</i> and <i>A. caulinodans</i>) or OD<sub>685</sub> (for <i>S. elongatus</i>) value of the lysate.</li>
<li>Measure the OD<sub>600</sub> (for <i>E. coli</i> and <i>A. caulinodans</i>) or OD<sub>685</sub> (for <i>S.
elongatus</i>) value of the lysate.</li>
<li>Perform the above operations once a day for about a week.</li>
</ol>
...
...
@@ -2023,8 +2064,14 @@
<divclass="h2">Co-culture of Immobilized Microorganisms</div>
</div>
<p><b>Reagent</b>: Initial <i>E .coli</i> bacterial fluid, Initial <i>S .elongatus</i> bacterial fluid, Initial <i>A .caulinodans</i> bacterial fluid, Sodium alginate powder, Calcium chloride, CoBG11-sucrose liquid medium and CoBG11-acetate liquid medium(see Experiments on <i>A. caulinodans</i>-Measurement of Growth Curves), Physiological saline, Milli-Q water</p>
acetate into 1 l Milli-Q water. Adjust the pH to 7.5.</li>
<li>Use the filter to filter the solution.</li>
</ol>
...
...
@@ -2044,10 +2092,14 @@
<p>Procedure:</p>
<ol>
<li>Centrifuge the microorganism fluid at 4000 rpm for 15 min. Remove the supernatant and weigh the wet weight.</li>
<li>For every 1 g cell, resuspend in 1 ml physiological saline, add 20 ml sodium alginate solution and stir until well combined.</li>
<li>Transfer the alginate-cell mixture into a small syringe (needle removed) and drips naturally into 20 ml 0.2 M calcium chloride solution. Meanwhile, stir with an electromagnetic stirrer.</li>
<li>Soak for 5 hours, then transfer the immobilized cells into CoBG11-PLUS medium and culture in a light shaker at 30℃, 130 rpm.</li>
<li>Centrifuge the microorganism fluid at 4000 rpm for 15 min. Remove the supernatant and weigh the wet weight.
</li>
<li>For every 1 g cell, resuspend in 1 ml physiological saline, add 20 ml sodium alginate solution and stir
until well combined.</li>
<li>Transfer the alginate-cell mixture into a small syringe (needle removed) and drips naturally into 20 ml 0.2
M calcium chloride solution. Meanwhile, stir with an electromagnetic stirrer.</li>
<li>Soak for 5 hours, then transfer the immobilized cells into CoBG11-PLUS medium and culture in a light shaker
at 30℃, 130 rpm.</li>
</ol>
<div>
...
...
@@ -2060,7 +2112,8 @@
<li>Transfer the immobilized cells into 1.5 ml EP tubes and weigh wet weight.</li>
<li>For every 1 g immobilized cell, add 1 ml 0.2 M phosphate buffer (pH=7.5).</li>
<li>Shake violently for 15 min until all solids dissolve.</li>
<li>Measure the OD<sub>600</sub> (for <i>E. coli</i> and <i>A. caulinodans</i>) or OD<sub>685</sub> (for <i>S. elongatus</i>) value of the lysate.</li>
<li>Measure the OD<sub>600</sub> (for <i>E. coli</i> and <i>A. caulinodans</i>) or OD<sub>685</sub> (for <i>S.
elongatus</i>) value of the lysate.</li>
<li>Perform the above operations once a day for about a week.</li>
</ol>
...
...
@@ -2068,14 +2121,21 @@
<divclass="h2">Intercellular Communication in Immobilized <i>E .coli</i> Through LuxR System</div>
<li>Centrifuge the microorganisms culture at 4000 rpm for 15 min, remove the supernatant and weigh wet weight.</li>
<li>For every 1 g cell, resuspend in 1 ml physiological saline, add 20 ml sodium alginate solution and stir until well combined.</li>
<li>Transfer the alginate-cell mixture into a small syringe (needle removed) and drips naturally into 20 ml 0.2 M calcium chloride solution. Meanwhile, stir with an electromagnetic stirrer.</li>
<li>Soak for 5 hours, then transfer the immobilized cells into CoBG11-sucrose medium and culture in a shaker at 37℃, 100 rpm.</li>
<li>Centrifuge the microorganisms culture at 4000 rpm for 15 min, remove the supernatant and weigh wet weight.
</li>
<li>For every 1 g cell, resuspend in 1 ml physiological saline, add 20 ml sodium alginate solution and stir
until well combined.</li>
<li>Transfer the alginate-cell mixture into a small syringe (needle removed) and drips naturally into 20 ml 0.2
M calcium chloride solution. Meanwhile, stir with an electromagnetic stirrer.</li>
<li>Soak for 5 hours, then transfer the immobilized cells into CoBG11-sucrose medium and culture in a shaker at
