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<h1>EXPERIMENTS</h1>
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<p>
Construct Plasmid and Search for Suitable Deep Eutectic Solvent
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</h2>
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<div class="accordion-body">
<h3>1. Obtain pet-22b ,BSLA and Bs2Est fragment by PCR</h3>
<figure>
<img alt="photo" class="w-40 mx-auto d-block" src="./images/fig0.png">
<img alt="photo" class="w-40 mx-auto d-block" src="./images/fig1.png">
<figcaption>
<center align="justify" style="font-size: 18px;"><b>Figure 1.</b>
The Enzyme digestion products are detected by agarose gel
electrophoresis, and the correct target fragment is 5476 bp. We obtained
the
correct
target fragment, and the sample is purified.
</center>
</figcaption>
</figure>
<div class="saut_1_ligne"></div>
<h3>2. Gibson assembly</h3>
<ol>
<li>Procedure:</li>
</ol>
<p style="text-indent: 2em;text-align: justify;"><b>a.</b> The above reaction
system was incubated
at 50 °C for 15 minutes
(inserting 1-2
fragments), 30 minutes (inserting 3 fragments simultaneously), or 60 minutes
(inserting 4-5 fragments simultaneously). If subsequent operations cannot be
carried
out immediately after the reaction is completed, the reaction sample can be
stored
at -20 °C. </p></br>
<ol>
<li>Transformation and cloning identification:</li>
</ol>
<p align="justify" style="text-indent: 2em;"><b>b.</b> Take 5uL of the reaction sample
from the
previous step and add it
to receptive
cells such as 50-100 uL of DH5a (note that the volume of the added DNA sample
should
not exceed 1/10 of the receptive volume). Gently mix and place the mixture on
ice
for 30 minutes.</p>
<p align="justify" style="text-indent: 2em;"><b>c.</b> Heat shock in a 42 °C water bath
for 90
seconds, then quickly return to the ice
bath and let stand for 3-5 minutes.
c. Add 500 uL of antibiotic free SOC or LB culture medium, gently mix well, and
shake
at 37 °C for 1 hour.</p>
<p align="justify" style="text-indent: 2em;"><b>d.</b> Centrifuge the bacterial solution
at
5000 g for 1 minute to precipitate the
bacterial body. Suck off most of the culture medium, leaving about 50-100 uL of
culture medium, and resuspend the bacterial body. Then apply all evenly onto LB
plates containing appropriate antibiotics and incubate overnight in a 37 °C
incubator.</br></p>
<p align="justify" style="text-indent: 2em;"><b>e.</b> The next day, select clones from
the
plate for colony PCR or extract plasmids
for
enzyme digestion identification, or directly select several clones for
sequencing
identification.</p>
<figure>
<img alt="photo" class="w-40 mx-auto d-block" src="./images/fig2.png">
<figcaption>
<center style="font-size: 18px;"><b>Figure 2.</b>
The pet-22b fragments and BSLA, Bs2Est are connected by Gibson assembly, and
the system is as follows.
</center>
</figcaption>
</figure>
<div class="saut_1_ligne"></div>
<figure>
<div class="saut_1_ligne"></div>
<img alt="photo" class="w-40 mx-auto d-block" src="./images/fig3.png">
<figcaption>
<center style="font-size: 18px;"><b>Figure 3.</b>
The pet-22b fragments and BSLA, Bs2Est are connected by Gibson assembly
</center>
</figcaption>
</figure>
<div class="saut_1_ligne"></div>
<h3>3. Selection of DES solutionR</h3>
<ol>
<li>Pre cultivation of microorganisms:</li>
</ol>
<p align="justify" style="text-indent: 2em;"><b>a.</b> Cultivation of Escherichia coli:
Use a needle to pick up WT and culture them in
shake
tubes with 150 μL amp-resistant LB liquid culture medium at 37 °C,900 rpm for 24
hours.</p>
<div class="saut_1_ligne"></div>
<ol>
<li>Main cultivation of microorganisms:</li>
</ol>
<p align="justify" style="text-indent: 2em;"><b>b.</b> Add 2 μL precultured bacterial
solution to 150 μL automatic induction culture
medium,
culture at 37 °C, 900 rpm for 16 hours.</p>
Preparation of DES solvent:
<p align="justify" style="text-indent: 2em;"><b>c.</b> TEA buffer:triethanolamine (50mM,
pH 7.4) </p>
<p align="justify" style="text-indent: 2em;"><b>d.</b> 95% choline chloride and ethylene
glycol (1:2): choline chloride 139.62g +
ethylene
glycol 124.14 g, heat and stir at 80 °C, and diluted to 95% with TEA buffer</p>
<p align="justify" style="text-indent: 2em;"><b>e.</b> 30% choline chloride and
acetamide (1:2): choline chloride 139.62 g + acetamide
118.14 g , heat and stir at 8 °C, and diluted to 30% with TEA buffer</p>
<p align="justify" style="text-indent: 2em;"><b>f.</b> 30% tetrabutylammonium bromide
and ethylene glycol (1:2): tetrabutylammonium
bromide
339.33 g + ethylene glycol 124.14 g , heat and stir at 80 °C, and diluted to 30%
with
TEA
buffer</p>
<p align="justify" style="text-indent: 2em;"><b>g.</b> 75% choline chloride and ethylene
glycol (1:2): choline chloride 139.62 g +
ethylene
glycol 124.14 g , heat and stir at 80 °C, and diluted to 75% with TEA buffer</p>
</p>
<figure>
<img alt="photo" class="w-80 mx-auto d-block" src="./images/fig4.png" style="width: 600px;">
<div class="saut_1_ligne"></div>
<figcaption>
<center style="font-size: 18px;"><b>Figure 4.</b>
Screening system of BSLA in three DESs.
</center>
</p>
</figcaption>
</figure>
<p align="justify">
(a)-(c) Residual activity of BSLA WT at different DES concentrations.</br>
(b)The
standard deviation to evaluate the applicability of the 96-well MTP-based
screening system for directed BSLA WT evolution. From left to right, the
compounds are 95% (v/v) ChCl:ethylene glycol, 30% (v/v) ChCl:acetamide, and
30% (v/v) TBPB:ethylene glycol, which used for the screening system to
obtain residual activity of 30-40% of the BSLA WT.</br>
<div class="saut_1_ligne"></div>
<figure>
<img alt="photo" class="w-80 mx-auto d-block" src="./images/fig5.png"style="width: 600px;>
<figcaption>
<center style="font-size: 18px;"><b>Figure 5.</b>
Determine the solution for the high-throughput screening system.
</center>
</figcaption>
</figure>
<p align="justify">After
excluding the DES types that tend to solidify at room temperature or are not
easy to handle with high viscosity, we used choline chloride and ethylene
glycol in a 1:2 ratio to configure concentrations of the solution.</p>
</div>
</div>
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<h2 class="accordion-header h2_exp">
<button class="accordion-button">
<p>Establish the BSLA and Bs2Est Mutant Library</p>
</button>
</h2>
<div class="accordion-collapse" id="item_2_collapse" style="display:none;">
<div class="accordion-body">
<h3>1. Extraction of plasmid DNA </h3>
<ol>
<li>Extracting bacterial DNA with a Axygen kit for extracting and
purifying DNA</li>
</ol>
<div class="saut_1_ligne"></div>
<h3>2. PCR</h3>
<ol>
<li> <a href="./images/Primers used for corner engineering of the BSLA gene in pet.pdf"
style="color:#589f7d;">Click Here</a> to obtain
primers used for corner engineering of the BLSA gene in pet-22</li>
</ol>
<div class="saut_1_ligne"></div>
<h3>3. Agarose gel electrophoresis </h3>
<ol>
<li>Preparation:</li>
</ol>
<p align="justify" style="text-indent: 2em;"><b>a.</b> 10X DNA Loading buffer</p>
<p align="justify" style="text-indent: 2em;"><b>b.</b> 15000 bp DNA Marker</p>
<p align="justify" style="text-indent: 2em;"><b>c.</b> PCR product</p>
<p align="justify" style="text-indent: 2em;"><b>d.</b> Nucleic acid gel dye(1 g agarose
+100mL 1X TAE)</p>
<p align="justify" style="text-indent: 2em;"><b>e.</b> Buffer solution for nucleic acid
gel electrophoresis()dilute 50X TAE to 1X with
<p align="justify" style="text-indent: 2em;"><b>f.</b> RO</p>
<div class="saut_1_ligne"></div>
<ol>
<li>Usage process:</li>
</ol>
<p align="justify" style="text-indent: 2em;"><b>a.</b> Add 100 mL 1X TAE to 1 g agar
powder
and add it into the conical flask specially
made
for agarose gel.</p>
<p align="justify" style="text-indent: 2em;"><b>b.</b> Boil in the microwave three times
until completely dissolved, cool until not too
hot
to touch (45-55 °C), add 10 uL of nucleic acid dye, shake gently, and pour into the
prepared mold, with a thickness of 60mm. Wait for it to solidify, then pull out the
comb
vertically to avoid damaging the sample hole.</p>
<p align="justify" style="text-indent: 2em;"><b>c.</b> Store the prepared nucleic acid
gel in the lunch box.</p>
<p align="justify" style="text-indent: 2em;"><b>e.</b> Add 1X TAE buffer solution to the
electrophoresis tank, and place the prepared gel
run
(large hole recovery gel and small hole verification gel, with the gel hole located
at
the negative electrode), with the buffer solution not passing through the sample
hole.</p>
<p align="justify" style="text-indent: 2em;"><b>f.</b> Sampling: Select the appropriate
size marker, mix the sample with the loading
buffer,
and then load the sample.</p>
<p align="justify" style="text-indent: 2em;"><b>g.</b> Set the voltage and press "run"
to
run the glue for about 30 minutes.</p>
<p align="justify" style="text-indent: 2em;"><b>h.</b> Take out the nucleic acid gel and
observe it with a light microscope.</p>
<div class="saut_1_ligne"></div>
<ol>
<li>Precautions:</li>
</ol>
<p align="justify" style="text-indent: 2em;"><b>a.</b> Pay attention to protection and
wear gloves when operating!</p>
<p align="justify" style="text-indent: 2em;"><b>b.</b> To make glue, dilute TAE should
be used, heated and cooled to 45 °C before adding
nucleic acid dye. The prepared glue can be stored at 4 °C for one week.</p>
<p align="justify" style="text-indent: 2em;"><b>c.</b> The adhesive runs from the
negative electrode to the positive electrode.</p>
<figure>
<img alt="photo" class="w-40 mx-auto d-block" src="./images/fig7.png">
<img alt="photo" class="w-40 mx-auto d-block" src="./images/fig8.png">
<img alt="photo" class="w-40 mx-auto d-block" src="./images/fig9.png">
<figcaption>
<center style="font-size: 18px;"><b>Figure 6.</b> A part of nucleic acid gel
</center>
</figcaption>
</figure>
<div class="saut_1_ligne"></div>
<h3>4. Transformation of receptive cells</h3>
<p align="justify" style="text-indent: 2em;"><b>a.</b> Take 100 µL of thawed receptive
cells on ice, add the target plasmid, gently mix
well, and let stand on ice for 30 minutes. </p>
<p align="justify" style="text-indent: 2em;"><b>b.</b> Heat shock the sample in a 42 °C
water bath for 45-60 seconds, transfer it
quickly to an ice bath, and let it stand for 2 minutes (do not shake the sample
during the process of standing on ice, otherwise the conversion efficiency will be
reduced). </p>
<p align="justify" style="text-indent: 2em;"><b>c.</b> Add 700 µL of sterile liquid
medium (SOB or LB) without antibiotics to the
centrifuge tube, mix well, and resuscitate at 37 °C and 200 rpm for 60 minutes. </p>
<p align="justify" style="text-indent: 2em;"><b>d.</b> According to the needs of the
experiment, draw different volumes of
resuscitation fluid and evenly spread it on the SOB or Lysogeny broth containing
corresponding antibiotics, and put the plate upside down in the 37 °C incubator for
overnight culture. </p>
<div class="saut_1_ligne"></div>
<h3>5. Select strains for pre cultivation</h3>
<p align="justify" style="text-indent: 2em;">The bacterial liquid was transferred to the
liquid LB supplemented with 1% 100
mg/mL Amp, at the same time.</br>
Pre cultivation system: 200 μL liquid LB + 1% 100 mg/mL Amp +bacterial broth
</p>
<div class="saut_1_ligne"></div>
<h3>6. Main culture</h3>
<p align="justify" style="text-indent: 2em;">2 μL pre cultured bacterial solution, add
self induced culture medium, culture them in
4 mL LB shake tubes with amp-resistant properties at 37 °C, 900 rpm for 16 hours.
</p>
<div class="saut_1_ligne"></div>
<h3>7. Freeze storage of glycerol bacteria</h3>
<p align="justify" style="text-indent: 2em;"><b>a.</b> Generally, the bacterial solution
used for seed preservation needs to be thicker,
so the cultivation time needs to be overnight;</p>
<p align="justify" style="text-indent: 2em;"><b>b.</b> Under the condition of Aseptic
technique, add 500 uL bacterial solution and 500 uL
50% glycerol solution into the sterilized 2 mL cryopreservation tube respectively,
mix gently and fully;</p>
<p align="justify" style="text-indent: 2em;"><b>c.</b> Glycerol cryopreserved bacteria
can be directly stored in a -80 °C
refrigerator.The frozen bacteria can be stored under this condition for several
years. Subsequent freezing and thawing operations will reduce the shelf life.</p>
<p align="left">(Note: Items that come into
contact with the bacterial solution, such as gun heads,
cryotubes, and 50% glycerol solution, must be in a sterile state and ensure that the
operation is carried out under sterile conditions.)</p>
<div class="saut_1_ligne"></div>
<h3>8. Enzyme activity measurement</h3>
<ol>
<li>The usage process of the enzyme-linked immunosorbent assay instrument</li>
</ol>
<p align="justify" style="text-indent: 2em;"><b>a.</b> Turn on the computer and enzyme
marker power;</p>
<p align="justify" style="text-indent: 2em;"><b>b.</b> Place the enzyme label plate
correctly. Do not put it in with a cover to prevent
it from getting stuck inside the instrument; If the sample is accidentally spilled,
it must be cleaned in a timely manner; Do not touch the card slot area when
transferring the enzyme label board to prevent hand injuries;</p>
<p align="justify" style="text-indent: 2em;"><b>c.</b> Open the software and set
parameters such as wavelength, temperature, and
oscillation frequency;</p>
<p align="justify" style="text-indent: 2em;"><b>d.</b> Select the scanning hole
position;</p>
<p align="justify" style="text-indent: 2em;"><b>e.</b> Conduct a scan, and the
instrument reading is relatively accurate between 0.4
and 1.0, with an upper limit of 4.0, if the value is too large, dilute it before
proceeding with the measurement; After scanning, save the data in a personal
folder;</p>
<p align="justify" style="text-indent: 2em;"><b>f.</b> Remove the enzyme marker board
and turn off the device.</p>
</div>
</div>
</div>
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<h2 class="accordion-header h2_exp">
<button class="accordion-button">
<p>The Mutants Obtained in the First Round were Rescreened for a Second Round</p>
</button>
</h2>
<div class="accordion-collapse" id="item_3_collapse" style="display:none;">
<div class="accordion-body">
<h3>1.Pre-work </h3>
<p align="justify" style="text-indent: 2em;"><b>a.</b> Pre-culture experiment content:
Toothpicks were dipped into glycerol bacteria in 96-well plates, in which four
parallel experiments were done for each mutant, and they were precultured with
LBamp. Take 150 μL from each well in the glycerol plate into the medium in
microtiter
plate, (37°C, 70% humidity, 900rpm, 24h) the obtained pre-culture solution can be
used for the subsequent main culture experiments.</p>
<p align="justify" style="text-indent: 2em;"><b>b.</b> The main culture experiment
content:
96-well plate as a container, take 2uL of pre-culture solution in the modified
auto-induced Amp medium (components) to express protein, (37 °C, 70% humidity, 900
rpm, 16 hours).</p>
<p align="justify" style="text-indent: 2em;"><b>c.</b> Enzyme activity assay:
The culture was centrifuged (4°C, 2000 rpm, 20 min) to obtain the crude enzyme,
which was used for further enzyme activity assay. The esterase activity of BSLA and
Bs2Est was determined using p-nitrophenyl butyrate (pNPB) as substrate. 95 μL
DES/Buffer and 5 μL WT(Variants)/EV were incubated for 2 h at room temperature, and
then the A410 nm was measured on the microtiter plate reader (Biotek Synergy HI,
USA), and the release of pNP was recorded in 8 minutes.</p>
<div class="saut_1_ligne"></div>
<h3>2.Week 1 </h3>
<figure>
<img alt="photo" class="w-80 mx-auto d-block" src="./images/fig10.png">
<figcaption>
<p align="justify">
<center style="font-size: 18px;"><b>Figure 7.</b> A total of 7positions
(P5/V6,V9/H10,A15/S16,K35/L36,T47/N48,S28/Q29,K88/N89,H76/S77)
were screened for beneficial mutations of BSLA. Beneficial variants have
been
preserved in glycerol 96-well plates in previous experiments and can be
used
for a
second round of screening.
</center>
</p>
</figcaption>
</figure>
<div class="saut_1_ligne"></div>
<h3>3.Week 2 </h3>
<figure>
<img alt="photo" class="w-80 mx-auto d-block" src="./images/fig11.png">
<figcaption>
<p align="justify">
<center style="font-size: 18px;"><b>Figure 8.</b> A total of 8
positions(L108/T109,S130/S131,M137/N138,I123/L124,I157/G158,Y161/S162/S163,L173/N174,L102/G103)
were screened for beneficial mutations of BSLA. Beneficial variants have
been preserved in glycerol 96-well plates in previous experiments and
can be
used for a second round of screening.
</center>
</p>
</figcaption>
</figure>
<div class="saut_1_ligne"></div>
<h3>4.Week 3 </h3>
<figure>
<img alt="photo" class="w-80 mx-auto d-block" src="./images/fig12.png">
<figcaption>
<p align="justify">
<center style="font-size: 18px;"><b>Figure 9.</b> A total of 8 positions
(P33-V34, E128-V129,
D155-N156,
K205-G206, M213-E214, M221-T222, E286-E287, and R38-F39) were screened
for
beneficial mutations of Bs2Est. Beneficial variants have been preserved
in
glycerol 96-well plates in previous experiments and can be used for a
second
round of screening.</center>
</p>
</figcaption>
</figure>
<div class="saut_1_ligne"></div>
<h3>5.Week 4 </h3>
<figure>
<img alt="photo" class="w-80 mx-auto d-block" src="./images/fig13.png">
<figcaption>
<p align="justify">
<center style="font-size: 18px;"><b>Figure 10.</b> A total of 7 positions
(F143-L144, S375-H376,
Q294-G295, R349-S350, R137-L138, L401-E402, and I421-T422) were screened
for
beneficial mutations of Bs2Est. The mutants were assayed for activity
according to the pre-culture, main culture and enzyme activity assay
methods
described previously, and the data were collated.
</center>
</p>
</figcaption>
</figure>
</div>
</div>
</div>
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<div class="accordion-item" id="item_4" onclick="growCircle(4)">
<h2 class="accordion-header h2_exp">
<button class="accordion-button">
<p>BSLA and Bs2Est Expression and Purification</p>
</button>
</h2>
<div class="accordion-collapse" id="item_4_collapse" style="display:none;">
<div class="accordion-body">
<h3>1.Week 1</h3>
<ol>
<li>Prepared the medium:</li>
</ol>
<p align="justify" style="text-indent: 2em;">LB-medium: Yeast extract 5 g/L, Tryptone
10 g/L, NaCl 10 g/L</p>
<p align="justify" style="text-indent: 2em;">Autoinduction medium:
The media include: media components, buffer components and induction components</p>
<figure>
<table class="center-table">
<tr>
<th>Composition</th>
<th>Weight</th>
</tr>
<tr>
<td>Peptone (aus Casein, Roth)</td>
<td>12 g</td>
</tr>
<tr>
<td>Yeast extract (Merck)</td>
<td>24 g</td>
</tr>
<tr>
<td>Glycerol (Roth)</td>
<td>5 g</td>
</tr>
<tr>
<td colspan="2">Add dd. H<span style="font-size: x-small;">2</span>O to 800
mL</td>
</tr>
</table>
<center style="font-size: 18px;"><b>Figure 11.</b> Media components,more mediums <a
href="images/mediums.pdf"
style="color: #0F5132;font-weight: 700;"><strong>Click
Here</strong></a></br>
</center>
</figure>
<div class="saut_1_ligne"></div>
<ol>
<li>Protein Induction Expression:</li>
</ol>
<figure>
<img alt="photo" class="w-40 mx-auto d-block" src="./images/fig15.png">
<div class="saut_1_ligne"> </div>
<img alt="photo" class="w-40 mx-auto d-block" src="./images/fig16.png">
<div class="saut_1_ligne"> </div>
<img alt="photo" class="w-40 mx-auto d-block" src="./images/fig17.png">
<figcaption>
<center align="justify" style="font-size: 18px;"><b>Figure 12.</b> SDS-PAGE.Based on
the protein gel analysis,
it can be
observed that both 24 hours and 48 hours of induction result in satisfactory
protein expression levels. Considering all factors, it is recommended to
choose a 24-hour induction period for optimal protein expression.
According to the gel plot analysis, BSLA was expressed in both LB and
self-induction medium, with auto-induction medium showing better expression.
Therefore, we chose the auto-induction medium to induce the expression of
BSLA.
</center>
</figcaption>
</figure>
<div class="saut_1_ligne"></div>
<ol>
<li>Purification:</li>
</ol>
Preparing Buffer Solutions with Different Imidazole Concentrations
<p align="justify" style="text-indent: 2em;"><b>a.</b> 50 mM potassium phosphate buffer
(pH 8.0)
1000 mL</p>
<p align="justify" style="text-indent: 2em;"><b>b.</b> 50 mM sodium phosphate buffer,
300 mM
sodium chloride, and 20 mM imidazole, pH
8.0</p>
<p align="justify" style="text-indent: 2em;"><b>c.</b> 50 mM sodium phosphate buffer,
300 mM
sodium chloride, and 50 mM imidazole, pH
8.0</p>
<p align="justify" style="text-indent: 2em;"><b>d.</b> 50 mM sodium phosphate buffer,
300 mM
sodium chloride, and 100 mM imidazole, pH
8.0</p>
<p align="justify" style="text-indent: 2em;"><b>e.</b> 50 mM sodium phosphate buffer,
300 mM
sodium chloride, and 250 mM imidazole, pH
8.0</p>
<p align="justify" style="text-indent: 2em;"><b>f.</b> 50 mM sodium phosphate buffer,
300 mM
sodium chloride, and 500 mM imidazole, pH
8.0</p><br>
Use Ni-NTA 6FF(Pre-Packed Gravity Column) to purified BSLA and Bs2Est. Selecting 250
mM Imidazole as the Final Elution Concentration
</p>
<div class="saut_1_ligne"></div>
<h3>2.Week 2</h3>
<ol>
<li>Inducing Expression of BSLA and Bs2Est Mutant in LB Medium</li>
</ol>
<p align="justify" style="text-indent: 2em;">Toothpick dip the bacterial liquid into 4
ml LB: First, use a
toothpick to transfer the bacterial liquid into a tube containing 4 mL of LB medium.
LB medium is a nutrient-rich medium commonly used for culturing bacteria such as
Escherichia coli. 37 °C, 220 rpm, 12 hours: Place the tube containing the bacterial
liquid in a constant temperature shaker and culture it at 37 °C and 220 rpm for 12
hours. This step aims to allow the bacterial liquid to
grow and proliferate under suitable temperature and agitation conditions. Inoculate
1% volume of the cultured liquid into 100 mL LB, 37 °C, 220 rpm, 2 hours: Take out
1%
(by volume) of the liquid cultured in the previous step and add it to a large
container containing 100 mL of auto-induction medium. Then, continue to culture it
at 30°C for 16h. </p>
<div class="saut_1_ligne"></div>
<ol>
<li>Toothpick dip the bacterial liquid into 4 ml LB</li>
</ol>
<p align="justify" style="text-indent: 2em;">First, use a toothpick to transfer the
bacterial liquid into a tube containing 4 mL
of LB medium. LB medium is a nutrient-rich medium commonly used for culturing
bacteria such as Escherichia coli. 37 °C, 220 rpm, 12 hours: Place the tube
containing
the bacterial liquid in a constant temperature shaker and culture it at 37 °C and
220 rpm for 12 hours. This step aims to allow the
bacterial liquid to grow and proliferate under suitable temperature and agitation
conditions. Inoculate 1% volume of the cultured liquid into 100 mL LB, 37 °C, 220
rpm,
2 hours: Take out 1% (by volume) of the liquid cultured in the previous step and add
it to a large container containing 100 mL of LB medium.</p>
<p align="justify" style="text-indent: 2em;">Then, continue to culture it
at 37 °C and 220 rpm for 2 hours. This step aims to
scale up the volume of the bacterial liquid to provide more cells for expression.
0.1 mM IPTG, 20 °C, 180 rpm, 24 hours: Add IPTG (Isopropyl
β-D-1-thiogalactopyranoside)
at a concentration of 0.1 millimolar to the culture medium to induce the expression
of the target protein. Next, continue to culture it at 20 °C and 180
rpm for 24 hours. IPTG is a synthetic compound that can mimic
signals bacteria receive in their natural environment, thus triggering the
expression of the target protein. E. coli cells were harvested by centrifugation at
11600 g for 10 min at 4 °C, and then washed twice with proper buffer (pH 7.5). The
washed cells were resuspended in 50 mM potassium phosphate buffer (pH 8.0) and
disrupted by sonication (10-sec pulse on, 15-sec pulse off, AMP 20%). </p>
<div class="saut_1_ligne"></div>
<figure>
<img alt="photo" class="w-40 mx-auto d-block" src="./images/fig18.png">
<figcaption>
<center style="font-size: 18px;"><b>Figure 13.</b> The picture on the left is
Bs2Est and the image on the
right is BSLA.
</center>
</figcaption>
</figure>
<div class="saut_1_ligne"></div>
<h3>3.Week 3-4</h3>
<ol>
<li>DESs resistance profiles and kinetic characterization of BSLA and Bs2Est
variants</li>
</ol>
<p align="justify" style="text-indent: 2em;">Pre-culture and master culture of all
beneficial mutants of BSLA and
Bs2Est. The expressed supernatant was taken for determination of different DES
concentrations. Different concentrations of DES solution are diluted with buffer
TAE. The method of measuring enzyme activity is consistent with the previous method
of constructing a mutant library.</p>
<p align="justify" style="text-indent: 2em;">K<span
style="font-size: x-small;">cat</span>/K<span
style="font-size: x-small;">M</span> assay with purified enzyme.
Configure different concentrations of PNP to
determine the 410 nm absorbance to plot the standard curve of PNP. All purified
mutants are assayed for protein concentration and protein standard curves are
plotted. Change the substrate pNPB concentration and monitor the absorbance value
after 5 min of the reaction. K<span style="font-size: x-small;">cat</span>/K<span
style="font-size: x-small;">M</span> is obtained by Graphpad.</p>
<figure>
<img alt="photo" class="w-80 mx-auto d-block" src="./images/fig19.png">
<figcaption>
<center style="font-size: 18px;"><b>Figure 14.</b> BSA concentration and PNP
</center>
</figcaption>
</figure>
<div class="saut_1_ligne"></div>
<ol>
<li>Tempreture and half life of BSLA and Bs2Est variants</br></li>
</ol>
<p align="justify" style="text-indent: 2em;">Pre-culture and master culture of all
beneficial mutants of BSLA and
Bs2Est. The expressed supernatant was taken for determination of different DES
concentrations. Different concentrations of DES solution are diluted with buffer
TAE. The method of measuring enzyme activity is consistent with the previous method
of constructing a mutant library.</p>
<p align="justify" style="text-indent: 2em;"> K<span
style="font-size: x-small;">cat</span>/K<span
style="font-size: x-small;">M</span> assay with purified enzyme.
Configure different concentrations of PNP to
determine the 410 nm absorbance to plot the standard curve of PNP. All purified
mutants are assayed for protein concentration and protein standard curves are
plotted. Change the substrate pNPB concentration and monitor the absorbance value
after 5 min of the reaction.K<span style="font-size: x-small;">cat</span>/K<span
style="font-size: x-small;">M</span> is obtained by Graphpad.</p>
</div>
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<p><b> References </b> <br><br></p>
<p>Sheldon, R. A.; Woodley, J. M. Role of Biocatalysis in Sustainable Chemistry. Chemical
Reviews 2017.
https://doi.org/10.1021/acs.chemrev.7b00203.</p><br>
<p>Cui, H.; Vedder, M.; Zhang, L.; Jaeger, K.-E.; Schwaneberg, U.; Davari, M. D. Polar Substitutions on the
Surface of a Lipase Substantially Improve Tolerance in Organic Solvents. ChemSusChem 2022, 15 (9),
e202102551. https://doi.org/10.1002/cssc.202102551.</p><br>
<p>Xu, P.; Liang, S.; Zong, M.-H.; Lou, W.-Y. Ionic Liquids for Regulating Biocatalytic Process: Achievements
and Perspectives. Biotechnology Advances 2021. https://doi.org/10.1016/j.biotechadv.2021.107702.</p><br>
<p>Abbott, A. P.; Capper, G.; Davies, D. L.; Rasheed, R. K.; Tambyrajah, V. Novel Solvent Properties of Choline
Chloride/Urea mixturesElectronic Supplementary Information (ESI) Available: Spectroscopic Data. See
Http://Www.Rsc.Org/Suppdata/Cc/B2/B210714g/. Chem. Commun. 2003, No. 1, 70-71.
https://doi.org/10.1039/b210714g.</p><br>
<p>Danait-Nabar, S.; Singhal, R. S. Investigation into the Chemical Modification of α-Amylase Using Octenyl
Succinic Anhydride: Enzyme Characterisation and Stability Studies. Bioprocess Biosyst Eng 2023, 46 (5),
645–664. https://doi.org/10.1007/s00449-023-02850-z.</p><br>
<p>Acevedo-Rocha, C. G.; Hoebenreich, S.; Reetz, M. T. Iterative Saturation Mutagenesis: A Powerful Approach to
Engineer Proteins by Systematically Simulating Darwinian Evolution. In Directed Evolution Library Creation;
Gillam, E. M. J., Copp, J. N., Ackerley, D., Eds.; Methods in Molecular Biology; Springer New York: New
York, NY, 2014; Vol. 1179, pp 103–128. https://doi.org/10.1007/978-1-4939-1053-3_7.</p><br>
<p>Taklimi, S. M.; Divsalar, A.; Ghalandari, B.; Ding, X.; Di Gioia, M. L.; Omar, K. A.; Saboury, A. A. Effects
of Deep Eutectic Solvents on the Activity and Stability of Enzymes. Journal of Molecular Liquids 2023, 377,
121562. https://doi.org/10.1016/j.molliq.2023.121562.</p><br>
<p> Wang, M.; Cui, H.; Gu, C.; Li, A.; Qiao, J.; Schwaneberg, U.; Zhang, L.; Wei, J.; Li, X.; Huang, H.
Engineering All-Round Cellulase for Bioethanol Production. ACS Synth. Biol. 2023, 12 (7), 2187–2197.
https://doi.org/10.1021/acssynbio.3c00289.</p><br>
<p> Qiao, J.; Sheng, Y.; Wang, M.; Li, A.; Li, X.; Huang, H. Evolving Robust and Interpretable Enzymes for the
Bioethanol Industry. Angew Chem Int Ed 2023, 62 (12), e202300320. https://doi.org/10.1002/anie.202300320.
</p><br>
<p> Cui, H.; Eltoukhy, L.; Zhang, L.; Markel, U.; Jaeger, K.; Davari, M. D.; Schwaneberg, U. Less Unfavorable
Salt Bridges on the Enzyme Surface Result in More Organic Cosolvent Resistance. Angew. Chem. Int. Ed. 2021,
60 (20), 11448–11456. https://doi.org/10.1002/anie.202101642.</p><br>
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