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experiments

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<!-- section 1 -->
<section class="m-b-6">
<div class="collapse-card" data-bs-toggle="collapse" data-bs-target="#collapse1" aria-expanded="false"
aria-controls="collapse">
<i>1. Primer Design</i>
</div>
<div class="collapse" id="collapse1">
<div class="card card-body">
<section>
<ol type="1">
<li>
Design primer using Snapgene with Helicobacter pylori, Salmonella Typhimurium, and Shigella Flexner’s
genome as templates. 10 templates will be designed in total. See the chart below for more details:
<div class="table-container m-t-2">
<table class="rw-65">
<tbody>
<tr>
<th>Bacteria Origin</th>
<th colspan="3">Number of primers</th>
</tr>
<tr>
<td></td>
<td>Primer-F</td>
<td>Primer-R</td>
<td>Target Sequence(s)</td>
</tr>
<tr>
<td><i>H. Pylori</i></td>
<td>2</td>
<td>2</td>
<td>cagA, 16S</td>
</tr>
<tr>
<td><i>S. Typhimurium</i></td>
<td>1</td>
<td>1</td>
<td>invA</td>
</tr>
<tr>
<td><i>S. flexneri</i></td>
<td>1</td>
<td>1</td>
<td>ipaH</td>
</tr>
<tr>
<td><i>Francisella tularensis</i> subsp. novicida FTG</td>
<td>1</td>
<td>1</td>
<td><i>FnCas12a</i></td>
</tr>
</tbody>
</table>
</div>
</li>
</ol>
<p>
See primer sequences as attached.
</p>
<p>
(<a href='http://parts.igem.org/Part:BBa_K4304021'>BBa_K4304021</a>; <a
href='http://parts.igem.org/Part:BBa_K4304022'>BBa_K4304022</a>; <a
href='http://parts.igem.org/Part:BBa_K4304023'>BBa_K4304023</a>; <a
href='http://parts.igem.org/Part:BBa_K4304024'>BBa_K4304024</a>; <a
href='http://parts.igem.org/Part:BBa_K4304025'>BBa_K4304025</a>; <a
href='http://parts.igem.org/Part:BBa_K4304026'>BBa_K4304026</a>; <a
href='http://parts.igem.org/Part:BBa_K4304027'>BBa_K4304027</a>; <a
href='http://parts.igem.org/Part:BBa_K4304028'>BBa_K4304028</a>; <a
href='http://parts.igem.org/Part:BBa_K4304029'>BBa_K4304029</a>; <a
href='http://parts.igem.org/Part:BBa_K4304030'>BBa_K4304030</a>)
</p>
</section>
</div>
</div>
</section>
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<!-- section 2 -->
<section class="m-b-6">
<div class="collapse-card" data-bs-toggle="collapse" data-bs-target="#collapse2" aria-expanded="false"
aria-controls="collapse">
<i>2. Plasmid construction by Genscript Biotech Company</i>
</div>
<div class="collapse" id="collapse2">
<div class="card card-body">
<section class="m-b-3">
<p>
See plasmid report from Genscript Biotech attached.
</p>
</section>
<!-- section 21 -->
<section class="m-b-6">
<div class="collapse-card card-sm" data-bs-toggle="collapse" data-bs-target="#collapse21"
aria-expanded="false"
aria-controls="collapse">
<i>2.1. 16S_pUC57 certificate</i>
</div>
<div class="collapse" id="collapse21">
<div class="card card-body">
<object type="application/pdf"
data="https://static.igem.wiki/teams/4304/wiki/experiments/t-ykpao-experiments-01.pdf"
style="width: 100%; height: 90vh;">
</object>
</div>
</div>
</section>
<!-- section 22 -->
<section class="m-b-6">
<div class="collapse-card card-sm" data-bs-toggle="collapse" data-bs-target="#collapse22"
aria-expanded="false"
aria-controls="collapse">
<i>2.2. ipaH_pUC57 certificate</i>
</div>
<div class="collapse" id="collapse22">
<div class="card card-body">
<object type="application/pdf"
data="https://static.igem.wiki/teams/4304/wiki/experiments/t-ykpao-experiments-02.pdf"
style="width: 100%; height: 90vh;">
</object>
</div>
</div>
</section>
<!-- section 23 -->
<section class="m-b-6">
<div class="collapse-card card-sm" data-bs-toggle="collapse" data-bs-target="#collapse23"
aria-expanded="false"
aria-controls="collapse">
<i>2.3. invA_pUC57 certificate</i>
</div>
<div class="collapse" id="collapse23">
<div class="card card-body">
<object type="application/pdf"
data="https://static.igem.wiki/teams/4304/wiki/experiments/t-ykpao-experiments-03.pdf"
style="width: 100%; height: 90vh;">
</object>
</div>
</div>
</section>
<!-- section 24 -->
<section>
<div class="collapse-card card-sm" data-bs-toggle="collapse" data-bs-target="#collapse24"
aria-expanded="false"
aria-controls="collapse">
<i>2.4. cagA_pUC57 certificate</i>
</div>
<div class="collapse" id="collapse24">
<div class="card card-body">
<object type="application/pdf"
data="https://static.igem.wiki/teams/4304/wiki/experiments/t-ykpao-experiments-04.pdf"
style="width: 100%; height: 90vh;">
</object>
</div>
</div>
</section>
</div>
</div>
</section>
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<!-- section 3 -->
<section class="m-b-6">
<div class="collapse-card" data-bs-toggle="collapse" data-bs-target="#collapse3" aria-expanded="false"
aria-controls="collapse">
<i>3. Transformation of the plasmid into BL21(DE3) E. coli</i>
</div>
<div class="collapse" id="collapse3">
<div class="card card-body">
<section>
<ol type="1" class="l-top-05 text-justify">
<li>Take out five 50μl <i>E. coli</i> BL21(DE3) competent cells from the -80℃ fridge and place them on ice.
Wait for
5 minutes until the competent cells unfreeze.
</li>
<li>In each of the five microtubes, add in 3μl 50ng/μl of the plasmids (16S_pUC57, ipaH_pUC57, invA_pUC57,
cagA_pUC57, Cas12a_pET-28a). Set it aside for 25 minutes.
</li>
<li>Place the microtube in a 42℃ water bath and heat shock for 45 seconds.</li>
<li>Place the tube back on the ice for 2 minutes.</li>
<li>Add 700μl lysogeny broth (LB) into each of the tubes and incubate at 200 rpm for 60 minutes.</li>
<li>Centrifuge at 5000 rpm for 2 minutes and coat on LB medium plates.</li>
</ol>
</section>
</div>
</div>
</section>
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<!-- section 4 -->
<section class="m-b-6">
<div class="collapse-card" data-bs-toggle="collapse" data-bs-target="#collapse4" aria-expanded="false"
aria-controls="collapse">
<i>4. Inoculate single colonies</i>
</div>
<div class="collapse" id="collapse4">
<div class="card card-body">
<section>
<ol type="1" class="l-top-05 text-justify">
<li>Plate all 5 <i>E. coli</i> strains on the LB medium plates, respectively (<i>Cas12a</i> strain on the
plate with Kana
antibiotics and the other 4 plates with Ampicillin antibiotics)
</li>
<li>incubate overnight and use a pipette to pick a single colony of <i>E. coli</i></li>
<li>inoculate single colonies in 3ml LB. A total of 5 flasks will be prepared, incubate overnight at 37℃,
200rpm.
</li>
</ol>
</section>
</div>
</div>
</section>
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<!-- section 5 -->
<section class="m-b-6">
<div class="collapse-card" data-bs-toggle="collapse" data-bs-target="#collapse5" aria-expanded="false"
aria-controls="collapse">
<i>5. Culture <i>E. coli</i></i>
</div>
<div class="collapse" id="collapse5">
<div class="card card-body">
<section>
<ol type="1" class="l-top-1 text-justify">
<li>
Prepare one 500 ml (to culture <i>Cas12a</i> <i>E. coli</i>）and three 300ml LB（To culture four colonies of GM <i>E. coli</i>
with different integrated template sequences*****, each trial requires 25ml, the excess will be on standby）
<br>
<span class="d-block m-t-1">Weigh the following reagents with an electronic balance and add them into 4
conical flasks using a
spatula
in the following sequence:</span>
<table class="rw-65 m-t-1 m-b-1">
<tbody>
<tr>
<td>Tryptone*</td>
<td>5 g</td>
<td>3 g</td>
</tr>
<tr>
<td>Yeast extract**</td>
<td>2.5 g</td>
<td>1.5 g</td>
</tr>
<tr>
<td>NaCl***</td>
<td>5 g</td>
<td>3 g</td>
</tr>
<tr>
<td>Suspend with ddH<sub>2</sub>O**** until the volume reaches</td>
<td>500 ml</td>
<td>300 ml</td>
</tr>
</tbody>
</table>
*Tryptone is used to provide essential amino acids such as peptides and peptones to the bacteria <br>
**Yeast Extract provides a range of organic compounds for bacteria growth <br>
***Sodium chloride balances osmotic pressure and provides sodium ions for the bacterium <br>
****ddH<sub>2</sub>O: double distilled water <br>
*****four types of template sequences：
<table class="rw-65 m-t-1">
<tbody>
<tr>
<th>Name of the sequence</th>
<th>Originates from</th>
</tr>
<tr>
<td>16S</td>
<td><i>Helicobacter pylori</i></td>
</tr>
<tr>
<td>cagA</td>
<td><i>Helicobacter pylori</i></td>
</tr>
<tr>
<td>invA</td>
<td><i>Salmonella typhimorium</i></td>
</tr>
<tr>
<td>ipaH</td>
<td><i>Shigella flexneri</i></td>
</tr>
</tbody>
</table>
</li>
<li>
Autoclave all LB (lysogeny broth)
<br>
*No need to adjust the pH as both sodium hydroxide and TRIS buffer are usually unnecessary.
</li>
<li>
Allocate 150ml autoclaved LB into two equal aliquots, each 75ml.
</li>
<li>
Add 75μl of Kan+ antibiotics into one flask, and 75μl of Amp+ antibiotics into the other (as the volume
ratio between LB and antibiotics should be 1000:1). Label the flasks.
</li>
<li>
Prepare 4 sets of cell culture tubes, each with 3 tubes.
</li>
<li>
In each group of tubes, allocate 5ml of Amp+ LB into one tube，and 5ml Kan+ LB into the other two.
</li>
<li>
Inoculate bacterium (Add 50μl of <i>E. coli</i>-containing-medium into each tube, ending up with four tubes with
four different types of GM <i>E. coli</i>), and label the date, its antibiotics, and integrated sequence on the
tube. The number and types of cell culture tubes are recorded in the table below:
<br>
<table class="rw-65 m-t-1">
<tbody>
<tr>
<th>Integrated sequence</th>
<th>Plasmid name</th>
<th>Corresponding antibiotics</th>
<th>Number of tubes</th>
</tr>
<tr>
<td><i>Cas12a</i></td>
<td>pET-28a</td>
<td>Kan+</td>
<td>8</td>
</tr>
<tr>
<td>16S</td>
<td rowspan="4">pUC57</td>
<td rowspan="4">Amp+</td>
<td>1</td>
</tr>
<tr>
<td>cagA</td>
<td>1</td>
</tr>
<tr>
<td>invA</td>
<td>1</td>
</tr>
<tr>
<td>ipaH</td>
<td>1</td>
</tr>
</tbody>
</table>
</li>
<li>
Place all tubes in the shaker rock for 16 hours, allow it to proliferate
</li>
</ol>
</section>
</div>
</div>
</section>
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<!-- section 6 -->
<section class="m-b-6">
<div class="collapse-card" data-bs-toggle="collapse" data-bs-target="#collapse6" aria-expanded="false"
aria-controls="collapse">
<i>6. Purify Cas12a proteins</i>
</div>
<div class="collapse collapse-6" id="collapse6">
<div class="card card-body">
<!-- section 61 -->
<section class="m-b-6">
<div class="collapse-card card-sm" data-bs-toggle="collapse" data-bs-target="#collapse61"
aria-expanded="false"
aria-controls="collapse">
<i>6.1 Transcription of Cas12a proteins in E. coli</i>
</div>
<div class="collapse collapse-61" id="collapse61">
<div class="card card-body">
<section>
<h3 style="margin-top: 1rem !important;"><u><i>
6.1.1 Transfer cas12a-containing E. coli for reproduction at a greater scale
</i></u></h3>
<span><u>Purpose</u>: For greater amount of <i>E. coli</i> cloning</span><br>
<span><u>Reagents</u>: LB, antibiotic K+, <i>Cas12a</i> containing <i>E. coli</i></span><br>
<ol type="1" class="l-top-05 m-t-2 text-justify">
<li>Prepare 3 conical flasks, each holding 150ml of LB medium</li>
<li>Add 150μl of antibiotic K+ into each flask using a pipette (note: add after the K+ solution fully
melts, as it was previously frozen)
</li>
<li>Add 5μl bacterial solution that contains <i>Cas12a</i> into each flask (while the normal ratio of
<i>E.
coli</i>
to LB is 1:1000, we increased the ratio to accelerate the speed of bacterial cloning at a greater
scale）
</li>
<li>Place the flasks into a shaker and incubate for 5 hours</li>
</ol>
</section>
<section>
<h3 style="margin-top: 1rem !important;" id="#612"><u><i>
6.1.2. Measure OD value of E. coli (dedicated to inducing Cas12a protein expression)
</i></u></h3>
<span><u>Purpose</u>: Check if the concentration is suitable for the addition of IPTG</span><br>
<span><u>Reagents</u>: LB, antibiotic K+, <i>Cas12a</i> containing <i>E. coli</i></span><br>
<ol type="1" class="l-top-05 m-t-2">
<li>Wipe off any residues on the Nanodrop from previous users with lens paper</li>
<li>Add 1μl of LB onto the probe using a pipette as the control group.</li>
<li>Run the test (by selecting the “nucleic acid” option)</li>
</ol>
</section>
<section>
<h3 style="margin-top: 1rem !important;"><u><i>
6.1.3. Addition of IPTG
</i></u></h3>
<span><u>Purpose</u>: Promote the transcription of <i>Cas12a</i> proteins in <i>E. coli</i> colonies in
different
IPTG
environments while also allowing comparison</span><br>
<span><u>Reagents</u>: LB, IPTG</span><br>
<ol type="1" class="l-top-05 m-t-2">
<li>Add 30μl of IPTG into flask 1 (containing 150ml LB and <i>E. coli</i>), creating a concentration of
0.2μM/μl
</li>
<li>Add 75μl of IPTG into flask 2 (containing 150ml LB and <i>E. coli</i>), creating a concentration of
0.5μM/μl
</li>
<li>Add 120μl of IPTG into flask 3 (containing 150ml LB and <i>E. coli</i>), creating a concentration of
0.8μM/μl
</li>
</ol>
</section>
</div>
</div>
</section>
<!-- section 62 -->
<section class="m-b-6">
<div class="collapse-card card-sm" data-bs-toggle="collapse" data-bs-target="#collapse62"
aria-expanded="false"
aria-controls="collapse">
<i>6.2. Cas12a protein extraction</i>
</div>
<div class="collapse" id="collapse62">
<div class="card card-body">
<section>
<h3 style="margin-top: 1rem !important;"><u><i>
6.2.1. Purify Cas12a Protein
</i></u></h3>
<ol type="1" class="l-top-05 m-t-2 text-justify">
<li>
Collect <i>E. coli</i> bacteria <br>
<span style="font-size: 16px"><u>Purpose</u>: to separate it from LB</span> <br>
<ol type="a" class="ol-mark mark-quota-alpha-left l-start">
<li>Centrifugation the bacteria medium at 4000 rpm</li>
<li>Discard the supernatant</li>
</ol>
</li>
<li>
Suspend <i>E. coli</i> within Buffer A <br>
<ol type="a" class="ol-mark mark-quota-alpha-left l-start">
<li>Add 5 ml of Buffer A into the sediments, allow the biomass to float</li>
</ol>
</li>
<li>
Ultrasound Cell Lysis of <i>E. coli</i><br>
<span style="font-size: 16px">
<u>Purpose</u>: Break the bacteria membranes so that <i>Cas12a</i> proteins can be extracted and
purified
</span> <br>
<ol type="a" class="ol-mark mark-quota-alpha-left l-start">
<li>Place the entire sample on ice (maintain a temperature of 4 °C to preserve its proteins) and
place it into an ultrasound cell crusher.
</li>
<li>Set the total time as 10 minutes and the interval as 5 seconds (to close for 5 seconds every
time the system worked for 5 seconds). The power ratio should be at 70%.
</li>
</ol>
</li>
<li>
Centrifugation of Ultrasound Results <br>
<ol type="a" class="ol-mark mark-quota-alpha-left l-start">
<li>Place the sample in the centrifuge. Set the system at 4000 rpm, 20 minutes, 4 °C.</li>
<li>After centrifugation, extract the supernatant (containing CAS 12a proteins)</li>
</ol>
</li>
</ol>
</section>
<section>
<h3 style="margin-top: 1rem !important;"><u><i>
6.2.2. Nickel affinity purification of Cas12a protein
</i></u></h3>
<span>
<u>Principle</u>: The 6×His tail of <i>Cas12a</i> protein is strongly attracted to the nickel column, therefore
will
not be washed down by Buffer A but will be washed down by Buffer B. Whereas other proteins, attach more
firmly or less firmly than the <i>Cas12a</i>, thus will be cleaned by adding Buffer A or will not be
washed by
Buffer B.
</span>
<ol type="1" class="l-top-05 m-t-2 text-justify">
<li>
Mix the reagents below in the assigned portion as shown in the chart to obtain 0.5 L His buffer A
<br>
<table class="m-t-1 m-b-1 rw-65">
<tbody>
<tr>
<td colspan="2">Buffer A: 0.5L</td>
</tr>
<tr>
<td>Ingredients</td>
<td></td>
</tr>
<tr>
<td>20 mM Na<sub>2</sub>HPO<sub>4</sub>.2H<sub>2</sub>O</td>
<td>1.78 g</td>
</tr>
<tr>
<td>500 mM NaCl</td>
<td>14.6 g</td>
</tr>
<tr>
<td>20 mM Imidazole</td>
<td>1.02 g</td>
</tr>
<tr>
<td colspan="2">HCl (6M) until pH reaches 7.4 and the volume reaches 0.5L</td>
</tr>
</tbody>
</table>
</li>
<li>
Mix the reagents below in the correct portion as mentioned in the chart below and obtain 0.25 His
buffer B
<br>
<table class="m-t-1 m-b-1 rw-65">
<tbody>
<tr>
<td colspan="2">Buffer B: 0.25L</td>
</tr>
<tr>
<td>Ingredient</td>
<td></td>
</tr>
<tr>
<td>20 mM Na<sub>2</sub>HPO<sub>4</sub>.2H<sub>2</sub>O</td>
<td>1.89 g</td>
</tr>
<tr>
<td>500 mM NaCl</td>
<td>7.3 g</td>
</tr>
<tr>
<td>500 mM Imidazole</td>
<td>8.5 g</td>
</tr>
<tr>
<td colspan="2">HCl (6M) until pH reaches 7.4 and the volume reaches 0.25L</td>
</tr>
</tbody>
</table>
</li>
</ol>
<ol type="1" start="1" class="l-top-05 text-justify">
<li>Complete installing the nickel affinity purification system by stacking the nickel column onto a
new microtube. Then, place it on ice.
</li>
<li>Using a pipette, add 4 times the nickel column’s size worth of Buffer A to moist the column.
Collect one tube of the solution under the nickel column and label it as the “first filter”.
</li>
<li>Add the supernatant into the nickel column. Decant slowly and gradually. Keep the velocity of
the flow at 1ml/min.
</li>
<li>Add 6 ml of Buffer A into the nickel column. Keep the velocity of the flow at 1ml/min.</li>
<li>Collect one tube of the solution under the nickel column and label it as the“last filter”.</li>
<li>Repeat step 4 for two more times.</li>
<li>Add 3 ml of Buffer B into the nickel column. Keep the velocity of the flow at 1ml/min.</li>
<li>Collect two tubes of the solution under the nickel column and label them as “<i>Cas12a</i>
protein #1”
and “<i>Cas12a</i> protein #2” respectively. They are the target protein.
</li>
<li>Add 2 ml of His Buffer B into the nickel column.</li>
<li>Collect one tube of the solution under the nickel column and label it as “Buffer B elution”.
</li>
<li>Add 5 ml of Buffer A into the nickel column.</li>
<li>Collect one tube of the solution under the nickel column and label it as “Buffer A elution”.
</li>
<li>Wash the nickel column by decanting 10ml of His buffer A and 5ml 20% ethanol</li>
</ol>
</section>
<section>
<h3 style="margin-top: 1rem !important;"><u><i>
6.2.3. Measure the concentration of Cas12a protein in the resultant solution
</i></u></h3>
<ol type="1" class="l-top-05 m-t-2 text-justify">
<li>Wash off any residue on the NanoDrop using ddH<sub>2</sub>O.</li>
<li>Add 2μl of Buffer B onto the probe using a pipette (As the negative control group). Record the
protein concentration. Wash off any residue on the NanoDrop.
</li>
<li>Add 2μl of “<i>Cas12a</i> protein #1” solution onto the probe using a pipette. Record the protein
concentration. Wash off any residue on the NanoDrop.
</li>
<li>Add 2μl of “<i>Cas12a</i> protein #2” solution onto the probe using a pipette. Record the protein
concentration. Wash off any residue on the NanoDrop.
</li>
</ol>
</section>
</div>
</div>
</section>
<!-- section 63 -->
<section>
<div class="collapse-card card-sm" data-bs-toggle="collapse" data-bs-target="#collapse63"
aria-expanded="false"
aria-controls="collapse">
<i>6.3. Protein electrophoresis of Cas12a proteins</i>
</div>
<div class="collapse" id="collapse63">
<div class="card card-body">
<section>
<span><u>Purpose</u>: To test the content and purity of the extracted <i>Cas12a</i> proteins</span><br>
<span><u>Material</u>: Protein 40μl, 4× Loading buffer 10μl, 7 microtubes</span><br>
</section>
<section>
<h3 style="margin-top: 1.5rem !important;"><u><i>
6.3.1 Protein Electrophoresis preparation (1mm thick mm gel)
</i></u></h3>
<ol type="1" class="l-top-05 m-t-2 text-justify">
<li>In a sterile cup, add 2ml of 2× gel solution A and 2ml of gel solution B</li>
<li>Using a pipette add 55μl TEMED, and gently swirl the cup until all reagents are fully mixed</li>
<li>Add the mixture gently, aware not to introduce any gas bubbles</li>
<li>Add ethanol using a pipette until the liquid level reaches the top</li>
<li>Wait for 6 to 10 minutes at room temperature, and allow the separating gel to solidify. After the
gel solidifies, dispose of the ethanol.
</li>
<li>Mix 0.75ml 2× upper gel solution A and 0.75ml 2× colored upper gel solution B (red) and 15μl TEMED
in a sterile cup, gently swirl the cup
</li>
<li>Gently pour the mixture between two glass plates (mold) and insert a comb on the top</li>
<li>Wait for 10-15 minutes until the stacking gel solidify.</li>
<li>Extract the comb.</li>
</ol>
</section>
<section>
<h3 style="margin-top: 1.5rem !important;"><u><i>
6.3.2. Protein electrophoresis of Cas12a proteins
</i></u></h3>
<ol type="1" class="l-top-05 m-t-2 text-justify">
<li>
Use a pipette to inject 40μl of protein into each of the seven microtubes in the order of nickel
column filter.
<br>
<ol type="i">
<li>first filtrate</li>
<li>last filtrate</li>
<li>Cas12a1</li>
<li>Cas12a2</li>
<li>Buffer B</li>
<li>Buffer A</li>
<li>Bacteria sediment (obtained from the residue in “purifying <i>Cas12a</i> proteins”)</li>
</ol>
</li>
<li>Add 10μl of 4× loading buffer into each of the seven microtubes</li>
<li>
Then put the tubes into a 100℃ metal bath for 10 minutes. This is to break the bonds in proteins and
disrupt their complex dimensional structures so that they will travel in a regular route during the
electrophoresis.
</li>
<li>
Make Tris-Glycine SDS buffer by mixing the following reagents. <br>
<ul style="list-style-type: disc">
<li>1 pack of Tris-Glycine SDS buffer powder</li>
<li>1L ddH<sub>2</sub>O</li>
</ul>
</li>
<li>Fill the middle cell in the protein electrophoresis machine with Tris-Glycine SDS buffer, and let it
evenly overflow into the two cells on the side. Make sure the water level in the outer cells exceeds
half of the container’s size.
</li>
<li>Make sure the positive and the negative electrodes are plugged in correctly.</li>
<li>Add 20μl of the sample protein (7 samples in total) into separate tubes carefully.</li>
<li>Add three markers at 20μl, to fill in all wells and ensure each tube has the same weight.</li>
<li>Start the electrolysis with a setting of 180V, for 45 minutes.</li>
<li>When the machine is turned on, air bubbles should travel upwards (opposite from the electrical
current).
</li>
<li>After the electrolysis, use Coomassie brilliant blue to stain the protein gel (that contains the
seven samples), which then visualizes the protein traces after destaining.
</li>
<li>Put the dyed protein gel onto the horizontal oscillator and incubate for 90 min.</li>
<li>Wash the protein gel using Coomassie brilliant blue decolorizing solution and soak it in the same
solution for 18 hours
</li>
<li>Place gel into GenoSens 2000 Gel Documentation and Analysis System to collect results.</li>
</ol>
</section>
<section>
<h3 style="margin-top: 1.5rem !important;"><u><i>
6.3.3. BCA test of Cas12a proteins’ concentration
</i></u></h3>
<ol type="1" class="l-top-05 m-t-2 text-justify">
<li>Dilute 20μl 5mg/ml BSA standard solution with 250μl DD water into a beaker to produce 0.04μg/ml BSA
standard protein solution
</li>
<li>In 9 of the wells on the lightproof microplate, add 100μl BCA reagent and 100μl standard protein
solution
</li>
<li>In 2 of the wells on the lightproof microplate, add 100μl BCA reagent and 100μl sample <i>Cas12a</i>
protein
solution
</li>
<li>Incubate the reagents in a gradient thermal cycler at 37 °C for 30 minutes.</li>
<li>Test using Subtract max</li>
</ol>
</section>
</div>
</div>
</section>
</div>
</div>
</section>
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<!-- section 8 -->
<section class="m-b-6">
<div class="collapse-card" data-bs-toggle="collapse" data-bs-target="#collapse8" aria-expanded="false"
aria-controls="collapse">
<i>8. Incubate sgRNA and Cas12a proteins and test the reaction system</i>
</div>
<div class="collapse" id="collapse8">
<div class="card card-body">
<section>
<u>Purpose</u>: Reassemble Cas12a-sgRNA compound and test if the system can recognize the target sequences in
the form
of plasmids, oligo DNAs and bacterium after lysis.
</section>
<!-- section 81 -->
<section class="m-b-6 m-t-3">
<div class="collapse-card card-sm" data-bs-toggle="collapse" data-bs-target="#collapse81"
aria-expanded="false"
aria-controls="collapse">
<i>8.1. Incubation of sgRNA and Cas12a proteins and test the resulting system using oligo DNA</i>
</div>
<div class="collapse" id="collapse81">
<div class="card card-body">
<section>
<h3 style="margin-top: 1rem !important;"><u><i>
8.1.1. Electrophoresis test
</i></u></h3>
<ol type="1" class="l-top-05 m-t-2 text-justify">
<li>
Prepare oligoDNA groups:
<table class="rw-100 m-t-1 m-b-2">
<tbody>
<tr>
<th colspan="6">Materials needed:</th>
</tr>
<tr>
<td></td>
<td>oligo DNA concentration /ng/μl</td>
<td>oligo DNA volume/μl</td>
<td>sgRNA volume/μl</td>
<td>cas12a protein volume/μl</td>
<td>reaction buffer volume/μl</td>
</tr>
<tr>
<td>ipaH</td>
<td>4.9</td>
<td>20.4</td>
<td>5</td>
<td>3</td>
<td>21.6</td>
</tr>
<tr>
<td>invA</td>
<td>64.95</td>
<td>1.54</td>
<td>5</td>
<td>3</td>
<td>40.46</td>
</tr>
<tr>
<td>16S</td>
<td>0.925</td>
<td>0.925</td>
<td>5</td>
<td>3</td>
<td>41.075</td>
</tr>
<tr>
<td>cagA</td>
<td>2.59</td>
<td>2.59</td>
<td>5</td>
<td>3</td>
<td>39.41</td>
</tr>
</tbody>
</table>
<p>
*Note that the volume of oligo DNA varies according to its concentration. But each centrifuge tube
should hold a total of 100M oligo DNA. The reaction buffer will also vary to achieve a fixed volume
of 50 μl.
</p>
<ol type="a" class="l-start-3">
<li>In 8 separate 200μL centrifuge tube, add in sgRNA (cagA sgRNA1, cagA sgRNA2, ipaH sgRNA1, ipaH
sgRNA2, invA sgRNA1, invA sgRNA2, 16S sgRNA1, 16S sgRNA2) cas12a protein and reaction buffer in
the following volumes using a pipette in a clean bench.
</li>
<li>Centrifuge the mixture for 30s.</li>
<li>Incubate the solution in a gradient thermal cycler at 37℃ for 10 minutes.</li>
<li>In each centrifuge tube, add in the corresponding oligo DNA according to the volume recorded in
the table above using a pipette.
</li>
<li>Centrifuge the systems briefly.</li>
<li>In 4 separate centrifuge tubes, add in 50μl of ipaH, invA, cagA ,and 16S oligo DNA without
incubation as the controlled group.
</li>
<li>Incubate all tubes in a gradient thermal cycler at 37℃ for 2 hours and then at 95℃ for 5 minutes
to denature <i>Cas12a</i> proteins.
</li>
</ol>
</li>
<li>
Electrophoresis of all 8 oligo DNA tubes:
<ol type="a" class="l-start-2">
<li>Add 2μl 10×Loading Buffer to each 20μl Oligo DNA system</li>
<li>Load 10μl 10× Buffer and Oligo DNA mixture into each well</li>
<li>Load 10μl DNA marker in the well</li>
<li>Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. This
will take about 20 minutes
</li>
<li>Open UV light to visualize DNA fragments</li>
</ol>
</li>
</ol>
</section>
</div>
</div>
</section>
<!-- section 82 -->
<section class="m-b-6">
<div class="collapse-card card-sm" data-bs-toggle="collapse" data-bs-target="#collapse82"
aria-expanded="false"
aria-controls="collapse">
<i>8.2. Incubation of sgRNA and Cas12a proteins and test the resulting system using plasmid</i>
</div>
<div class="collapse" id="collapse82">
<div class="card card-body">
<section>
<h3 style="margin-top: 1rem !important;"><u><i>
8.2.1. Using ssDNA (fluorescent) and multiscan ascent
</i></u></h3>
<ol type="1" class="l-top-05 m-t-2 text-justify">
<li>
Prepare plasmids groups:
<table class="rw-100 m-t-1 m-b-2">
<tbody>
<tr>
<th colspan="6">Materials needed:</th>
</tr>
<tr>
<td></td>
<td>plasmid vol/μL</td>
<td>ssdna volume/μL</td>
<td>sgRNA volume/μL</td>
<td>cas12a protein volume/μL</td>
<td>reaction buffer volume/μL</td>
</tr>
<tr>
<td>ipaH</td>
<td>0.5</td>
<td>3</td>
<td>5</td>
<td>3</td>
<td>38.5</td>
</tr>
<tr>
<td>invA</td>
<td>0.5</td>
<td>3</td>
<td>5</td>
<td>3</td>
<td>38.5</td>
</tr>
<tr>
<td>16S</td>
<td>0.5</td>
<td>3</td>
<td>5</td>
<td>3</td>
<td>38.5</td>
</tr>
<tr>
<td>cagA</td>
<td>0.5</td>
<td>3</td>
<td>5</td>
<td>3</td>
<td>38.5</td>
</tr>
</tbody>
</table>
<ol type="a">
<li>In 8 separate 200μL centrifuge tubes, add in sgRNA (cagA I, cagA II, ipaH I, ipaH II, invA I,
invA II, 16S I , 16S II), cas12a protein and reaction buffer in the volumes depicted in the above
chart using a pipette in a clean bench.
</li>
<li>Incubate the solution in a gradient thermal cycler at 37℃ for 10 minutes.</li>
<li>In each centrifuge tube, add in the corresponding plasmids and ssDNA according to the volume
recorded in the table above.
</li>
<li>Incubate the tubes in a gradient thermal cycler at 37℃ for 2 hours and then at 95℃ for 5 minutes
to denature <i>Cas12a</i> proteins.
</li>
</ol>
</li>
<li>
Configure Negative Control Group of Cas12A-crRNA complex (without incubation):
<ol type="a" class="l-start-2">
<li>Add 38.5μl reaction buffer and 3μl of 50nM Cas12a to a microtube.</li>
<li>Divide the reagent into 8 micro tubes of 5μl each (the excess will be used as a backup)</li>
<li>5μl of corresponding sgRNA (cagA sgRNA1, cagA sgRNA2, invA sgRNA1, invA sgRNA2, 16S sgRNA1, 16S
sgRNA2, ipaH sgRNA1, ipaH sgRNA2) were added into each tube
</li>
<li>0.5μl of corresponding plasmid and 3μl ssDNA fluorescent probe were added in every tube</li>
</ol>
</li>
<li>
Measurement of ssDNA fluorescence value:
<ol type="a" class="l-start-2">
<li>Pipette 37 μl of solution from each of the tubes and controlled group* into the 96 wells light
screen
</li>
<li>Measure fluorescence response of the bacteria culture using Multiskan Ascent</li>
</ol>
</li>
</ol>
</section>
</div>
</div>
</section>
<!-- section 83 -->
<section>
<div class="collapse-card card-sm" data-bs-toggle="collapse" data-bs-target="#collapse83"
aria-expanded="false"
aria-controls="collapse">
<i>8.3. Incubation of sgRNA and Cas12a proteins and test the resulting system using <i>E. coli</i> culture</i>
</div>
<div class="collapse" id="collapse83">
<div class="card card-body">
<section>
<h3 style="margin-top: 1rem !important;"><u><i>
8.3.1. Test the efficacy of Cas12a-sgRNA system using electrophoresis:
</i></u></h3>
<ol type="1" class="l-top-05 m-t-2 text-justify">
<li>
Bacteria lysis
<ol type="a">
<li>Using a pipette, transfer 1 ml of each of the four <i>E. coli</i> cultures(16S, cagA, ipaH,
invA) into
a centrifuge tube and centrifuge for 1 minute at 8000 rpm. Discard the supernatant.
</li>
<li>Add 50 μLllysis buffer into all four tubes.</li>
<li>Place all tubes into a water bath and heat at 80℃ for 10 minutes</li>
<li>Centrifuge all tubes again at 8000 rpm for 2 minutes</li>
<li>Pipette at least 5 μl for each of the four <i>E. coli</i> colonies into at least two separate 20
μl
microtubes. A total of 14 tubes of the bacterium are collected in this step.
</li>
<li>Extract 10 μl of each of the four <i>E. coli</i> inoculants into a microtube using a pipette and
store
them in a 4°C environment to use for electrophoresis in step 4.
</li>
</ol>
</li>
<li>
Incubation of <i>Cas12a</i> and sgRNA system and cleavage of target sequences
<ol type="a" class="l-start-2">
<li>
A total of 16 1.5ml microtubes (1 tube for each sgRNA and the other as the controlled group)
will be prepared each with 45μl of solution. Add all the following reagents and mediums using a
pipette and on a clean bench.
<ul style="list-style-type: disc">
<li><i>Cas12a</i> protein (3 μl)</li>
<li>50/500ng sgRNA (5μl)</li>
<li>Reaction buffer (37 μl)</li>
</ul>
</li>
<li>Incubate in a gradient thermal cycler at 37℃ for 10 minutes.</li>
<li>In all 14 microtubes, add in 5 μl of each of the four corresponding bacteria and label the
tubes.
</li>
<li>Place one complete set of tubes with non-repetitive sgRNA sequences (8 tubes) in a gradient
thermal cycler and PCR at 12000×g for 5 minutes.
</li>
<li>Centrifuge the tube at 8000×g, allowing the bacterium fully interact with the system.</li>
<li>Incubate the product at 37℃ for 2 hours.</li>
<li>Further heat the tubes at 95 °C for 5 minutes to halt all denature all <i>Cas12a</i> proteins
and halt all protein activities.
</li>
</ol>
</li>
<li>
PCR
<ol type="a" class="l-start-2">
<li>Add 60 μl Mix Master Enzyme, 42 μl ddH2O, 12 μl Primer (6 μl Forward Primer and 6 μl Reverse
Primer) and 6 μl incubated medium from step 2 into a centrifuge tube for each of the 14 tubes and
run PCR in a gradient thermal cycler.
</li>
</ol>
</li>
<li>
Prepare electrophoresis
<ol type="a" class="l-start-2">
<li>
Add in the following reagents using a pipette into a beaker and briefly heat it until all reagents
fully dissolve
<ul style="list-style-type: disc">
<li>2 g Agarose Regular</li>
<li>100 ml TAE solution</li>
<li>3 μl Nucleic Acid Gel Stain</li>
</ul>
</li>
<li>Add gel into the mould and wait until it solidifies.</li>
<li>Add TAE buffer and gel into the electrophoresis machine</li>
</ol>
</li>
<li>
Pipette 10 μl of 18 bacterial samples into each of the gel’s wells and run electrophoresis at 180V for
10 minutes.
</li>
</ol>
</section>
<section>
<h3 style="margin-top: 1rem !important;"><u><i>
8.3.2. Test the efficacy of the Cas12a-sgRNA system using ssDNA (fluorescent) and multiscan ascent
</i></u></h3>
<ol type="1" class="l-top-05 m-t-2 text-justify">
<li>
Bacteria lysis
<ol type="a">
<li>Using a pipette, transfer 1 ml of each of the four <i>E. coli</i> cultures into two centrifuge
tubes
and centrifuge for 1 minute at 8000 rpm. Discard the supernatant.
</li>
<li>Add 50 μl lysis buffer into all four tubes.</li>
<li>Place all tubes into a water bath and heat at 80℃ for 10 minutes</li>
<li>Centrifuge all tubes again at 8000 rpm for 2 minutes</li>
<li>Obtain 2 μl of the supernatant and pipette 4 μL for each of the two <i>E. coli</i> colonies into
two
separate 20 μl microtubes.
</li>
</ol>
</li>
<li>
Incubation of <i>Cas12a</i> and sgRNA system and target sequence cleavage
<ol type="a" class="l-start-2">
<li>
A total of 16 1.5ml microtubes (2 tubes for each plasmid and 8 as controlled) will be prepared
each with 50μl of solution. Add all the following reagents and mediums using a pipette and in a
clean bench.
<ul style="list-style-type: disc">
<li><i>Cas12a</i> protein (3 μl)</li>
<li>50/500ng sgRNA (5μl)</li>
<li>Reaction buffer (34 μl)</li>
</ul>
</li>
<li>Incubate in a gradient thermal cycler at 37℃ for 10 minutes.</li>
<li>To 8 of the 1.5 ml microtubes, add in 2 μl of the corresponding bacterium supernatant from step
1. Leave the other tubes bacteria-free.
</li>
<li>Incubate all tubes at 37℃ for 2 hours in a gradient thermal cycler.</li>
<li>Further heat all tubes at 95℃ for 5 minutes to halt protein activities.</li>
</ol>
</li>
<li>
Test using ssDNA and multiscan accent
<ol type="a" class="l-start-2">
<li>
In a 96 well light screen, add 3 μl of fluorescent ssDNA and 2 μl of 3 ipaH <i>E. coli</i>
plasmids (1
well for each of the two sgRNAs and 1 well without the plasmid as control), filling a total of 8
wells. In the four wells that are not the controlled group, add in <i>Cas12a</i>-sgRNA assay until
the
volume meets 50 μL.
</li>
<li>
Test for fluorescent using the multiscan accent every 0.5 hours for the next 2 hours. Results
are depicted in the table and graph below.
</li>
</ol>
</li>
</ol>
</section>
</div>
</div>
</section>
</div>
</div>
</section>
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<!-- section 9 -->
<section class="m-b-6">
<div class="collapse-card" data-bs-toggle="collapse" data-bs-target="#collapse9" aria-expanded="false"
aria-controls="collapse">
<i>9. Conduct LAMP</i>
</div>
<div class="collapse" id="collapse9">
<div class="card card-body">
<ol type="1" class="l-top-05 text-justify">
<li>
Unfreeze 2× Lamp Master mix and primer on ice and mix the two reagents thoroughly.
</li>
<li>
Create the reaction system by adding the following reagents in their assigned portions into a microtube.
<table class="rw-100 m-t-1">
<tbody>
<tr>
<th>Reagent</th>
<th>25μl Reaction system</th>
<th>Final concentration</th>
</tr>
<tr>
<td>2×Lamp Master Mix</td>
<td>12.5 μl</td>
<td>1×</td>
</tr>
<tr>
<td>FIP (10μM)</td>
<td>2μl</td>
<td>0.8μM</td>
</tr>
<tr>
<td>BIP (10μM)</td>
<td>2μl</td>
<td>0.8μM</td>
</tr>
<tr>
<td>Loop F (10μM)</td>
<td>1μl</td>
<td>0.4μM</td>
</tr>
<tr>
<td>Loop B (10μM)</td>
<td>1μl</td>
<td>0.4μM</td>
</tr>
<tr>
<td>F3 (10μM)</td>
<td>0.5μl</td>
<td>0.2μM</td>
</tr>
<tr>
<td>B3 (10μM)</td>
<td>0.5μl</td>
<td>0.2μM</td>
</tr>
<tr>
<td>Template DNA</td>
<td>25-100 ng</td>
<td>1-4 ng/μl</td>
</tr>
<tr>
<td>DNA polymerase</td>
<td>0.5μl</td>
<td>0.16 U/μl</td>
</tr>
<tr>
<td>Sterilized ddH2O</td>
<td>Up to 25μl</td>
<td>-</td>
</tr>
</tbody>
</table>
</li>
<li>
Place the microtube into a 65C water bath and heat it for 30-60 minutes.
</li>
<li>
Place the tube in a gradient thermo cycler and heat at 80C for 10 minutes to denature all DNA polymerase
</li>
</ol>
</div>
</div>
</section>
\ No newline at end of file
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......@@ -6,13 +6,20 @@
{% block page_content %}
<div class="page">
<div class="container">
<div class="article">
<div class="article text-justify">
<h1 class="content-header2">Experiments</h1>
<section>
<h2></h2>
<p></p>
</section>
<!-- section -->
{% include 'experiments/section-1.html' %}
{% include 'experiments/section-2.html' %}
{% include 'experiments/section-3.html' %}
{% include 'experiments/section-4.html' %}
{% include 'experiments/section-5.html' %}
{% include 'experiments/section-6.html' %}
{% include 'experiments/section-7.html' %}
{% include 'experiments/section-8.html' %}
{% include 'experiments/section-9.html' %}
</div>
</div>
</div>
......
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