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<h2 class="section-heading mb-4">
<span class="section-heading-lower"><strong>Experiments</strong></span>
</h2>
<span id="s1" class="section-heading-upper h3"><br />LB Preparation</span>
<ol>
<li>Take one bottle of BeyoPure™ LB Broth (Catalog No. ST156), open the package, and add
it entirely into a 1L blue-capped bottle.</li>
<li> Add ultrapure water from the Milli-Q® Type 1 Ultrapure Water System until it reaches
500 ml, shake well, apply autoclave tape, and slightly loosen the bottle cap.
</li>
<li>Sterilize at 121°C for 30 minutes.
</li>
</ol>
<span id="s1" class="section-heading-upper h3"><br />LB Agar Plate Preparation</span>
<ol>
<li>Take one bottle of BeyoPure™ LB Broth with Agar (Catalog No. ST158), open the package,
and add it entirely into a 1L blue-capped bottle.</li>
<li>Add ultrapure water from the Milli-Q® Type 1 Ultrapure Water System until it reaches 500
ml, shake well, apply autoclave tape, and slightly loosen the bottle cap. </li>
<li>Sterilize at 121°C for 30 minutes.</li>
<li>Allow it to cool to around 60°C, then add the corresponding antibiotic.</li>
</ol>
<span id="s1" class="section-heading-upper h3"><br />Stock Solution Preparation</span>
<ol>
<li>NaCl
<p>Weigh 1 g of NaCl into a 50 ml centrifuge tube, add ultrapure water from the Milli-Q®
Type 1 Ultrapure Water System until it reaches 50 ml, vortex to mix, let it stand
for 3 minutes, and filter sterilize using a sterile membrane filter (Merck
Millex™-GS Sterile Syringe Filter Unit, MCE, 0.22 μm). Prepare a 100 mM NaCl
solution and aliquot it into 2 ml EP tubes for storage.</p>
</li>
<li>MgCl2·6H2O
<p>Weigh 4.2 g of MgCl2·6H2O into a 50 ml centrifuge tube, add ultrapure water from the
Milli-Q® Type 1 Ultrapure Water System until it reaches 50 ml, vortex to mix, let it
stand for 3 minutes, and filter sterilize using a sterile membrane filter (Merck
Millex™-GS Sterile Syringe Filter Unit, MCE, 0.22 μm). Prepare a 200 mM Mg2+
solution and aliquot it into 2 ml EP tubes for storage.</p>
</li>
<li>KCl
<p>Using a pipette, take 5 ml of a 1M KCl solution, add ultrapure water from the
Milli-Q® Type 1 Ultrapure Water System until it reaches 50 ml, vortex to mix, let it
stand for 3 minutes, and filter sterilize using a sterile membrane filter (Merck
Millex™-GS Sterile Syringe Filter Unit, MCE, 0.22 μm). Prepare a 200 mM Mg2+
solution and aliquot it into 2 ml EP tubes for storage.</p>
</li>
<li>CaCl2
<p>Using a pipette, take 5 ml of a 1M CaCl2 solution, add ultrapure water from the
Milli-Q® Type 1 Ultrapure Water System until it reaches 50 ml, vortex to mix, let it
stand for 3 minutes, and filter sterilize using a sterile membrane filter (Merck
Millex™-GS Sterile Syringe Filter Unit, MCE, 0.22 μm). Prepare a 100 mM Ca2+
solution and aliquot it into 2 ml EP tubes for storage.</p>
</li>
<li>ZnSO4·7H2O
<p>Weigh 2.87 g of ZnSO4·7H2O into a 50 ml centrifuge tube, add ultrapure water from the
Milli-Q® Type 1 Ultrapure Water System until it reaches 50 ml, vortex to mix, let it
stand for 3 minutes, and filter sterilize using a sterile membrane filter (Merck
Millex™-GS Sterile Syringe Filter Unit, MCE, 0.22 μm). Prepare a 200 mM Zn2+
solution and aliquot it into 2 ml EP tubes for storage.</p>
</li>
<li>Kanamycin Stock Solution
<p>Weigh 1 g of kanamycin powder (Beyotime: ST101) and add it into a 50 ml centrifuge
tube, add 20 ml of Sterilized ddH2O (Sangon Biotech: B541017), vortex to mix, let it
stand for 3 minutes, and filter sterilize using a sterile membrane filter (Merck
Millex™-GS Sterile Syringe Filter Unit, MCE, 0.22 μm). Prepare a 50 mg/ml kanamycin
stock solution and aliquot it into 2 ml EP tubes for storage.</p>
</li>
<li>Chloramphenicol Stock Solution
<p>Weigh 600 mg of chloramphenicol powder (Beyotime: ST2722-1g) and add it into a 50 ml
centrifuge tube, add 20 ml of Sterilized ddH2O (Sangon Biotech: B541017), vortex to
mix, let it stand for 3 minutes, and filter sterilize using a sterile membrane
filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE, 0.22 μm). Prepare a 30
mg/ml streptomycin stock solution and aliquot it into 2 ml EP tubes for storage.</p>
</li>
</ol>
<span id="s1" class="section-heading-upper h3"><br />Plasmid Extraction from Single
Colonies</span>
<ol>
<li>All plasmid extraction reagents are used with the Tiangen Plasmid Mini Kit (DP103).
</li>
<li>Retrieve the glycerol stock of the target plasmid from the -80°C freezer, streak it onto
an appropriate antibiotic plate, and incubate overnight at 37°C.
</li>
<li>Pick a single colony from the plate and inoculate it into 5 ml of LB with the
appropriate antibiotic, incubating overnight at 220 rpm.
</li>
<li>Extract the plasmid using the Tiangen Plasmid Mini Kit.
</li>
<li>Measure the plasmid concentration using NanoDrop One.</li>
</ol>
<span id="s1" class="section-heading-upper h3"><br />Plasmid Extraction from a Library</span>
<ol>
<li>Retrieve the glycerol stock of the target library from the -80°C freezer, streak it onto
an appropriate antibiotic plate, and incubate overnight at 37°C. Thaw the remaining
bacterial suspension and transfer it into 100 ml of LB with the appropriate antibiotic,
incubating for 6 hours.
</li>
<li>Extract the plasmid library according to the instructions in the Tiangen Plasmid Mini
Kit (DP103), and measure the library concentration using NanoDrop One.</li>
<li>Pick 10 single colonies from the plate and perform Sanger sequencing for sampling to
verify the accuracy of the library.</li>
</ol>
<span id="s1" class="section-heading-upper h3"><br />Bacterial Transformation</span>
<ol>
<li>Retrieve Trans 5a Chemically Competent Cells (TransGene CD201-01) from the -80°C freezer
and place them on ice to thaw for 10 minutes.
</li>
<li>Add 10 ng of the target plasmid to the thawed competent cells, gently mix, and incubate
on ice for 30 minutes. Meanwhile, prepare a water bath at 42°C.
</li>
<li>Heat shock the mixture of competent cells and plasmid at 42°C for 45 seconds, then
immediately transfer the tube to ice for 2 minutes without shaking the centrifuge tube.
</li>
<li> Add 500 µl of sterile LB medium (without antibiotics) to each tube, mix well, and
incubate at 37°C with shaking at 200 rpm for 1 hour to allow bacterial recovery.
Meanwhile, prepare the appropriate antibiotic LB plates and place them in the 37°C
incubator.
</li>
<li>Based on the experimental requirements (plasmid or recombinant ligation product
transformation), pipette different volumes of transformed competent cells onto LB agar
plates containing the appropriate antibiotic and spread evenly. Place the plates in the
incubator at 37°C until the liquid is absorbed, then invert the plates and incubate
overnight at 37°C.
</li>
<li>Store the plates at 4°C after the colonies have grown.</li>
</ol>
<span id="s1" class="section-heading-upper h3"><br />Fluorescent kinetic assay</span>
<p>3 single colonies of Section I or Section II strains were inoculated into 0.9 ml LB medium
with specific antibiotics in a 2-ml 96-well deep-well plate (NEST), The plates were sealed
with breathable Nunc Seals (Thermo Scientific) and incubated at 37 °C and 1000 r.p.m.
overnight. The overnight cultures were diluted 1:300 into 900 µl LB medium with antibiotics
in 2-ml 96-well deep-well plates (NEST); the plates were sealed with Nunc Seals and
incubated at 37 °C and 1,000 r.p.m. metal ions were then added to achieve the desired ion
concentrations in the final system for our experimental conditions. After mixing by
pipetting, 135 µl cultures of each were transferred into 3 black 96-Well Cell Culture Plates
with Clear Bottom(In Vitro Scientific) respectively to set up replicates, another 15 ul
mineral oil were added in case of evaporation. The kinetic of fluorescence readings (515 nm
excitation wavelength and 555nm emission wavelength) were measure overnight in every 10
minutes on Spark® Multimode microplate Reader.
<span id="s1" class="section-heading-upper h3"><br />Library construction</span>
<p>
Plasmid library was assembled in one step. Promoter library were inserted into Transcription
1 plasmid in pSB1C3 sequentially with BsaI-HF®v2(NEB),rCutsmart,and T4 DNA Ligase（NEB）using
the program: 37 °C for 5 min, 16 °C for 5 min, 60 cycles.
</p>
<table class="table table-striped table-bordered table-hover">
<thead>
<tr>
<th>item</th>
<th>Volume(ul)</th>
</tr>
</thead>
<tbody>
<tr>
<td>Promoter library</td>
<td>3</td>
</tr>
<tr>
<td>Vector Plasmid</td>
<td>3</td>
</tr>
<tr>
<td>T4 Ligase buffer</td>
<td>2</td>
</tr>
<tr>
<td>rCutsmart</td>
<td>2</td>
</tr>
<tr>
<td>BsaI-HF®v2(NEB)</td>
<td>1.5</td>
</tr>
<tr>
<td>T4 DNA Ligase（NEB）</td>
<td>1.5</td>
</tr>
<tr>
<td>ddH2O</td>
<td>7</td>
</tr>
<tr>
<td>Total</td>
<td>20</td>
</tr>
</tbody>
</table>
<p>The resulting products were cleaned up using TIANquick Midi Purification Kit(TIANGEN) and
then transformed to 100 µl DH5a Chemically Competent Cell (TransGen) and then plated on
plates containing LB + 2% agar and grown overnight at 37 °C. Plates were stored at 4 °C
after growth.</p>
<span id="s1" class="section-heading-upper h3"><br />Fine-tuning cricuit screening assay</span>
<p>96 single colonies from Library construction were inoculated into 0.9 ml LB medium with
specific antibiotics in a 2-ml 96-well deep-well plate (NEST), The plates were sealed with
breathable Nunc Seals (Thermo Scientific) and incubated at 37 °C and 1000 r.p.m. overnight.
The overnight cultures were diluted 1:300 into 900 µl LB medium with antibiotics in 2-ml
96-well deep-well plates (NEST); the plates were sealed with Nunc Seals and incubated at 37
°C and 1,000 r.p.m. After 3 h, Cultures were diluted 1:100 into 900 µl LB medium with
antibiotics in 2-ml 96-well deep-well plates (NEST), and ion solutions were then added to
achieve the corresponding Cu(II) concentrations. After mixing by pipetting, 135 µl cultures
of each were transferred into 3 black 96-Well Cell Culture Plates with Clear Bottom(In Vitro
Scientific) respectively to set up replicates, another 15 ul mineral oil were added in case
of evaporation. The kinetic of optical density at 600 nm (OD600) and fluorescence readings
(515 nm excitation wavelength and 555nm emission wavelength) were measure overnight in every
10 minutes on Spark® Multimode microplate Reader. We inscribed the maximum fluorescence
values at each of the two concentrations and calculated the corresponding fluorescence
multiplicity.</p>
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