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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page_content %}
{% block header_content %}
<img class="sub-header-logo" src="https://static.igem.wiki/teams/4515/wiki/banner.jpg" />
{% endblock %}
<div class="row mt-4">
<div class="col-lg-8">
<h2>What should this page contain?</h2>
<hr>
<p>Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.</p>
<p>If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the <a href="http://parts.igem.org/Main_Page">Registry</a>.</p>
</div>
<div class="col-lg-4">
<h2>Inspirations</h2>
<hr>
<ul>
<li><a href="https://2019.igem.org/Team:Nantes/Experiments">2019 Nantes</a></li>
<li><a href="https://2019.igem.org/Team:TU_Eindhoven/Experiments">2019 TU Eindhoven</a></li>
<li><a href="https://2019.igem.org/Team:Mingdao/Demonstrate">2019 Mingdao</a></li>
<li><a href="https://2020.igem.org/Team:Amsterdam/Experiments">2020 Amsterdam</a></li>
<li><a href="https://2020.igem.org/Team:NCTU_Formosa/Experiments">2020 NCTU Formosa</a></li>
<li><a href="https://2020.igem.org/Team:USAFA/Experiments">2020 USAFA</a></li>
</ul>
{% block page_content %}
<div class="sub-page-bg safety-bg">
<div class="sub-page-content">
<div class="content-title">Experiments</div>
<div class="title blue-title accordion-item first-collapse" data-bs-toggle="collapse" data-bs-target=".content1"
aria-expanded="true">A. Plasmid construction of pLY15-opt </div>
<div class="sub-title accordion-item collapse content1" data-bs-toggle="collapse" data-bs-target="#content1-1"
aria-expanded="false">1. Take the ATCC824 genome as the template, then thl, hbd, crt, and ter genes were amplified
by PCR using thl, hbd, crt and ter primers. </div>
<div class="article-content collapse" id="content1-1">
▶ Amplification<br />
All reagents were melted at room temperature, mixed by shaking, centrifuged at 6000 rpm for 10 sec, and prepared
according to the following system:<br /><br />
<table class="sub-table margin-bottom-16" border="1" cellspacing="0" cellpadding="0">
<tr>
<th>Name</th>
<th>Amount added</th>
<th>Final concentration</th>
</tr>
<tr>
<td>2×TOROBlue Flash KOD DyeMix</td>
<td>25 μL</td>
<td>1×</td>
</tr>
<tr>
<td>10 μM upstream primer</td>
<td>1.5 μL</td>
<td>0.3μM</td>
</tr>
<tr>
<td>10 μM downstream primer</td>
<td>1.5 μL</td>
<td>0.3μM</td>
</tr>
<tr>
<td>DNA template </td>
<td>5 μL </td>
<td>GenomeDNA10-200 ng; Plasmid1-50ng; cDNA ~750 ng; Living specimen, crude extract 5 μL</td>
</tr>
<tr>
<td>ddH2O</td>
<td>Add to 50 μL</td>
<td></td>
</tr>
</table>
▶ The PCR program<br /><br />
<table class="sub-table" border="1" cellspacing="0" cellpadding="0">
<tr>
<th>Step</th>
<th>Temperature</th>
<th>Time</th>
<th>Numbers of circulation</th>
</tr>
<tr>
<td>Pre-denaturation</td>
<td>98℃</td>
<td>2 min </td>
<td></td>
</tr>
<tr>
<td>Denaturation</td>
<td>98°C</td>
<td>10 sec</td>
<td rowspan="3">25-45</td>
</tr>
<tr>
<td>Annealing</td>
<td>(Tm-5) °C </td>
<td>5 sec</td>
</tr>
<tr>
<td>Extension</td>
<td>68°C</td>
<td>1-10 sec/kb</td>
</tr>
</table>
</div>
<div class="sub-title accordion-item collapse content1" data-bs-toggle="collapse" data-bs-target="#content1-2"
aria-expanded="false">2. Agarose gel electrophoresis detection</div>
<div class="article-content collapse" id="content1-2">
▶ Prepare agarose gel: add 100 ml 1×TAE buffer into the conical flask, weigh 1.0 g agarose into a conical flask,
heat it in the microwave oven until it is completely melted, take it out and shake it well, then it is 1% agarose
gel.<br />
▶ Add Dye: when agarose gel is cooled to 70°C, add 10 uL nucleic acid dye, and mix well.<br />
▶ Preparation of gel board: waterproof tape is pasted on both ends of the glue bed to form an 8 mm high retaining
wall, press the tape, and place it on the horizontal table. Insert the comb, and the lower end of the comb is
about 1mm away from the bottom of the plate. Pour the 60℃ gel into the glue bed continuously, 3-4 mm high, avoid
bubbles, and solidify at room temperature. After complete solidification, tear off the adhesive tape, place it in
the electrophoresis tank, add the electrophoresis buffer, which is 1-2 mm higher than the glue surface, gently
pull out the comb obliquely from one end to remove the bubbles generated.<br />
▶ Sampling: add 5 μL DNA maker first, and then take 10 μL PCR reaction system for sample addition.<br />
▶ Electrophoresis: cover the electrophoresis tank, the sampling hole is located at the negative pole of the
electric field, and connects the power supply, 120V. When the front edge of the dye moves to the bottom edge for
1-2 mm, about 25-30 min, the electrophoresis ends.<br />
▶ Observation results: turn off the power, take out the gel, put it in the UV detector (324 nm), and verify if
there is a band of target DNA.
</div>
<div class="sub-title accordion-item collapse content1" data-bs-toggle="collapse" data-bs-target="#content1-3"
aria-expanded="false">3. Gel extraction. </div>
<div class="article-content collapse" id="content1-3">
<img class="w-80" src="https://static.igem.wiki/teams/4515/wiki/t-east-china-experiment01.jpg" /><br />
▶ 1. After DNA electrophoresis, quickly cut the gel containing the target DNA fragment under UV light. It is
recommended to blot the surface liquid of the gel with a paper towel and chop it, and try to remove the excess
gel. The gel was weighed (removing the empty tube weight) and 100mg of gel was equivalent to 100 μL volume, which
was used as a gel volume.<br />
▶ 2. Add an equal volume of Buffer GDP. 50 ~ 55 '℃ water bath for 7-10min, adjust the time appropriately according
to the gel size to ensure that the gel block is completely dissolved. Water bath period upside down mixing 2 times
to accelerate sol. (Adding 1-3 times the volume of A Buffer GDP does not affect the DNA recovery rate. If the DNA
fragment less than or equal to 100 bp is recovered, add 3 times the volume of Buffer GDP, add the same volume of
isopropanol after water bath sol, mix and then proceed to the third step.)<br />
▶ 3. Droplets on the wall of the tube were collected by brief centrifugation. The FastPure DNA Mini columns-G
adsorption column was placed in the Collection Tubes2ml Collection tube, and ≤700 μL sol solution was transferred
to the adsorption column, and centrifuged at 12,000 rpm (13,400×g) for 30-60 sec. If the sol volume is greater
than 700 μL, put the adsorption column back in the collection tube, and the remaining sol solution is transferred
to the column, 12,000 rpm (13,400×g) centrifugation 30-60 sec.<br />
▶ 4. Discard the filtrate and place the column in the collection tube. Add 300 μL Buffer GDP to the column.
Centrifugation at 12,000 rpm(13,400×g) for 30-60 sec.<br />
▶ 5. Discard the filtrate and place the adsorption column in the collection tube. Add 700 μL buffer GW (absolute
ethanol has been added) to the column. Centrifugation at 12,000rpm (13,400×g) for 30-60 sec. (Please add Buffer GW
around the wall of the column, or mix the cover upside down for 2-3 times after adding Buffer GW to help
completely wash the salt attached to the tube wall.)<br />
▶ 6. Repeat Step 5. (Two flushes with Buffer GW can ensure that the salt coating is completely removed,
eliminating the impact on subsequent experiments.)<br />
▶ 7. Discard the filtrate and put the adsorption column back into the collection tube. Centrifuge at 12,000 rpm
(13,400×g) for 2 min.<br />
▶ 8. Place the column in a 1.5 ml sterilized centrifuge tube, add 20-30 μL Elution Buffer to the center of the
adsorption column, and place for 2 min. Centrifuge at 12,000 rpm (13,400×g) for 1 min. The column was discarded
and the DNA was stored at -20℃. (If maximum yield is required, it is recommended that the resulting solution be
readded to the centrifugal adsorption column and step 8 is repeated for secondary elution. When recovering
fragments larger than 3kb, it is recommended to preheat the Elution Buffer to 55 ° C to improve the recovery
efficiency.)
</div>
<div class="sub-title accordion-item collapse content1" data-bs-toggle="collapse" data-bs-target="#content1-4"
aria-expanded="false">4. One-step cloning. </div>
<div class="article-content collapse" id="content1-4">
▶ Recombinant plasmids were transformed into competent cells, and LB plates were screened by colony PCR.<br />
▶ Preparation of selective medium plate: LB liquid medium is prepared with 15g agar powder /1000ml LB solid
medium. After sterilization, it is cooled to below 55 ℃ and erythromycin is added. The final concentration is 300
μg/ mL, and immediately falls plate in disposable Petri dishes.<br />
▶ Incubation on Ice: add recombinant plasmids into 50 μl prepared competent cells, mix well, and keep on ice for
30 minutes.<br />
▶ Heat Shock: Put competent cells into 42℃ water bath for 42 seconds.<br />
▶ Cooling: take it out and quickly keep it on ice for 2-3min.<br />
▶ Resuscitation: 1 ml non-resistant LB liquid medium was added to each tube, 37℃, 150 rpm, 45 min.<br />
▶ 5000rpm, 2min, suck and discard 700ul supernatant, resuspend the transformed competent cells or control samples
in the medium and coat them to the Petri dishes containing ampicillin respectively.
</div>
<div class="sub-title accordion-item collapse content1" data-bs-toggle="collapse" data-bs-target="#content1-5"
aria-expanded="false">5. The culture dish is turned upside down and incubated at 37℃ overnight. </div>
<div class="article-content collapse" id="content1-5"></div>
<div class="sub-title accordion-item collapse content1" data-bs-toggle="collapse" data-bs-target="#content1-6"
aria-expanded="false">6. Pick monoclonal culture seed solution. </div>
<div class="article-content collapse" id="content1-6"></div>
<div class="sub-title accordion-item collapse content1" data-bs-toggle="collapse" data-bs-target="#content1-7"
aria-expanded="false">7. Plasmid extraction</div>
<div class="article-content collapse" id="content1-7">
▶ Add absolute ethanol to rinsing solution PWT.<br />
▶ Centrifugate the overnight culture of bacteria at 12,000 rpm (~ 13,400 × g) for 1 min and remove the
supernatant.<br />
▶ Add 150 μL solution P1 to the centrifuge tube to resuspend bacterial precipitation.<br />
▶ Add 150 μl solution P2 into the centrifuge tube, and the tubes were gently turned up and down for 6-8
times.<br />
▶ Add 350 μL solution P3 into the centrifuge tube, immediately and quickly mix up and down for 12-20 times, 12,000
rpm (~13,400 × g) centrifuge for 2 min.<br />
▶ The supernatant collected in the previous step is transferred to the adsorption column CP3 with a pipette.
12,000 rpm (~ 13,400 × g) was centrifuged for 30 sec, the waste liquid in the collecting pipe was poured out, and
the adsorption column CP3 was put into the collecting pipe.<br />
▶ 300 mL of rinsing solution PWT was added into the column CP3, and 12,000 rpm (~ 13,400 × g) was centrifuged for
30 sec. Discard the filtrate and reuse the collection tube.<br />
▶ The adsorption column CP3 was put into the collection tube, and centrifuged at 12,000 rpm (~ 13,400 × g) for 1
min. Discard the filtrate and reuse the collection tube.<br />
▶ Place the adsorption column CP3 in a clean centrifuge tube and add 50-100μL elution buffer TB drops to the
middle of the adsorption membrane, The plasmid solution was collected into a centrifuge tube after centrifugation
at 12, 000 rpm (~13400 × g) for 30 seconds.
</div>
<div class="title blue-title accordion-item first-collapse" data-bs-toggle="collapse" data-bs-target=".content2"
aria-expanded="false">B. Preparation of natural competence </div>
<div class="article-content collapse content2">
1. Cultivate pIB184: LB medium (resistance: Erythromycin), 37° C, 220 rpm overnight. The plasmid was extracted the
next day.<br />
2. Culture of Streptococcus Brevis ATCC367: Glycerol-preserving bacteria were isolated at 37° C for 24 ~ 48 h, and
single colonies were cultured in the MRS medium overnight. Overnight culture of 2 ml was seeded into 50 ml GMRS
(Mixed with 1% glycine) and incubated at 37 ° C until OD600 reached ~ 0.6. The cells were collected by
centrifugation at 4 ° C, 4000 rpm, 10 min. The supernatant was removed and the cell particles were washed twice
with 1 mM MgCl2. Cells were then suspended in 0.5 ml SM buffer (952 mM sucrose and 3.5 mM MgCl2), and 100μL of
aliquots were stored at -80 ° C.
</div>
<div class="title blue-title accordion-item first-collapse" data-bs-toggle="collapse" data-bs-target=".content3"
aria-expanded="false">C. Electroporation and Inoculation of pIB184 and streptococcus Brevis ATCC367</div>
<div class="article-content collapse content3">
1. Plasmids were transformed into Streptococcus Brevis ATCC367 by electroporation.<br />
2. During electroporation, competent cells were mixed with DNA (about 1 µ g, 5 µ L). Incubated on ice for 5min,
and then transferred to a pre-cooled 2mm electric rotating cup for electroporation under the conditions of 2kV,
25UF,200 Ω. <br />
3. After electric shock, add 1ml fresh broth (add 500 mM sucrose, 20 mM MgCl2 and 2 mM CaCl2 to MRS), and
resuscitate at 37 ° C and 220rpm for 2~3h. <br />
4. After that, the cells were inoculated in medium containing antibiotics and incubated at 37 ° C for 24~48h.
</div>
<div class="title blue-title accordion-item first-collapse" data-bs-toggle="collapse" data-bs-target=".content4"
aria-expanded="false">D. Butanol fermentation and product detection </div>
<div class="article-content collapse content4">
1. After electroporation, the isolated colonies were cultured in MRS overnight and added with antibiotics. <br />
2. Transfer 0.7 ml of overnight cultured medium to 70 ml of deoxy-MRS and seeded into an anaerobic chamber for
overnight incubation.<br />
3. After culture, the cells were removed from the anaerobic chamber and incubated at 25 ° C for about 24 hours.
<br />
4. The cell density was measured with a DU730 photometer. Butanol and ethanol were detected by gas chromatography.
</div>
<div class="title blue-title accordion-item first-collapse" data-bs-toggle="collapse" data-bs-target=".content5"
aria-expanded="false">E. Extraction and detection of intracellular metabolites</div>
<div class="article-content collapse content5">
1. The cells were cultured at 25 ° C, and 70 ml of fermentation broth was filtered by 0.45 μm filter. The
metabolic reaction of the sample on a filter using methanol: Acetonitrile: WATER = 40:40:20 was cooled at -4℃ for
20 min. <br />
2. The samples were then transferred to a tube containing a quarter volume of 0.1 mm SiO2 dioxide beads and ground
three times for 3 minutes at 2,000 rpm. <br />
3. The supernatant was collected by centrifugation at 12,000 rpm for 30 min at 4℃. The above extraction steps are
repeated, and the supernatant is merged and filtered prior to metabolite detection. <br />
4. Metabolites were analyzed using High-performance liquid chromatography, UPLC and Q Exactive Orbitrap mass
spectrometer. The chromatographic column is T3 column (2.1×150 mM, 3 μm, Waters) , mobile phase A was 10 mM
ammonium acetate, mobile phase B was 100% acetonitrile, flow rate was 0.2 ml/min, elution gradient of Phase B was
0 min, 0% ; 1 min, 0% ; 1.1 min, 5% ; 7 min, 25% ; 8 min, 40% ; 11 min, 40% ; 11. 1min,0%；15 min,0%.<br />
5. Full scans were performed at M/z 700-1300 using electrospray ionization (ESI) in a positive ion mode, and all
acquired data were analyzed with XCalibur 4.0
</div>
</div>
</div>
{% endblock %}
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