Latest revision as of 03:54, 28 October 2020

Experiments

Cloning

For cloning of the fusion of single-chain variable fragment (scFv) and fragment crystallizable (Fc), the amino acid sequence of anti-HER2 scFv described by Ahmadzadeh, M.^[1] and the CH2 & CH3 region of immunoglobulin heavy constant gamma 1, which is the constant region of immunoglobulin heavy chains (UniProtKB-P01857), was used respectively. In addition, the hinge region of immunoglobulin heavy constant gamma 1 was used as the linker between scFv and Fc, a FLAG tag was fused to the C-terminus as well. Furthermore, we employed codon optimization to maximize the expression of a functional protein in Escherichia coli. The gene was synthesized by GenScript Biotech Corporation (Nanjing, CN). The gene was subcloned into gene expression vector pET19b, introduced a 10× histidine tag at the N-terminus.

Figure 1. Vector of scFv-Fc, pET19b.

For cloning of the fusion of mamC (GeneBank: AVM72836.1) and ZZ (GeneBank:M74186.1 ), the gene was synthesized and codon optimized by GenScript Biotech Corporation (Nanjing, CN). The gene was subcloned into gene expression vector pGEX-2TK, introduced a GST tag at the N-terminus.

Figure 2. Vector of mamC_ZZ, pGEX-2TK.

Expression of scFv-Fc

After the DNA sequence was confirmed (SUNYA, Zhejiang, CN), the pET19b was transformed into Escherichia coli SHuffle^®T7 Express Cells. The cells were grown in LB medium at 37°C until the OD600 reached 0.6, at which time protein expression was induced by adding filter sterilized isopropyl-β-D-thiogalactopyranoside (IPTG). The cells were then incubated at 30°C for an additional time to expression. Afterwards, the cells were harvested by centrifugation at 4000 rpm for 12 min. The cell pellet was resuspended in lysis buffer (50 mM Tris/HCl, pH 7.5, 500 mM NaCl, 0.1% CA630, 10% glycerol and 5 mM β-mercaptoethanol) and the cells were lysed by sonication. The cell extraction was clarified by centrifugation at 10000 rpm for 15 min.

Expression of mamC-ZZ

After the DNA sequence was confirmed (SUNYA, Zhejiang, CN), the pGEX-2TK construct was transformed into BL21 (DE3). The process of growth, induction and harvest was the same as that of scFv-Fc. The cell pellet team was resuspended in lysis buffer (50 mM Tris-HCl pH8.0 5 mM EDTA and 1.5 mM NaCl, phenylmethylsulfonyl fluoride). Add lysozyme (Solarbio, Beijing, CN) to a final concentration of 500 μg/mL, incubating for 30 min at room temperature and centrifuging at 12000 rpm for 30 seconds. Discard the supernatant to remove the periplasm protein. Add RIPA IV (Sangon, Shanghai, CN) and incubate on ice for 10 min, vibrating the tubes for several times during the incubation. The cell extract was clarified by centrifugation at 10000 rpm for 10 min.

Purification of scFv-Fc

The protein was purified by immunoprecipitation (IP) using anti‐DYKDDDDK G1 affinity resin (GenScript, Nanjing, CN). Resuspend the resin to form a uniform slurry and transfer 40 μL of the slurry into a 1.5 mL vial. Add 500 μL TBS into the vial and gently mix the resin, centrifuge at 6000 rcf for 30 seconds, and remove supernatant carefully. Repeat this step for three times. Add 1 mL supernatant of the cell lysate to the washed resin. Mix by end‐to‐end rotation on a tube rotator overnight at 4°C. To remove the nonspecific binding segment, centrifuge at 6000 rcf for 30 seconds, carefully remove supernatant and add 500 μL TBS to wash the resin three times. Remove as much supernatant as possible without disturbing the resin, add 120 μL of acid elution buffer (0.1 M glycine HCl, pH 3.5) into the washed resin and use a wide bore pipette tip to gently resuspend the resin. Incubate at room temperature for 5 minutes, mix gently by tapping the tube once or twice during the incubation period. After incubation, centrifuge at 6000 rcf for 30 seconds. Carefully transfer supernatant into a new vial containing 5 μL 1 M Tris, pH 9.0 for further application.

Optimum Concentration of IPTG for Expressing

The transformed cells were grown until the OD600 reached 0.6, at which time protein expression was induced by a gradient from 0 to 2 mM of IPTG. The cells were then incubated at 30°C for an additional 24 h.

Optimum Induction Time for Expressing

The transformed cells were grown until the OD₆₀₀ reached 0.6, at which time protein expression was induced by 2 mM IPTG. The cells were then incubated at 30°C for a gradient from 0 to 24 h. For negative control, none inducer was introduced.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS–PAGE)

The cell lysate or purified protein was mixed 1:1 (v:v) with 2× loading buffer (TIANGEN, Beijing, CN), heated for 5 min at 95°C and subjected to SDS–PAGE (5% stacking gel, 8% separating gel). Gels were run at 110V for 15vmin and then at 160V for ~50 min in running buffer.

Coomassie Staining

Following SDS–PAGE, gels was stained with Coomassie brilliant blue (0.1% Coomassie brilliant blue R-250, 25% isopropanol, 10% acetic acid) for 2 h at room temperature. Destain in destaining buffer (5% ethanol, 10% acetic acid) overnight.

Western Blotting of Proteins

Following SDS–PAGE, samples were transferred to nitrocellulose membrane (Merck) by semi-dry transfer (Bio-Rad). Membranes were blocked 1 h at room temperature in 5% (w/v) nonfat milk in TBST, probed with a rabbit anti-FLAG antibody (Abcam) overnight at 4°C, washed with TBST, probed with a goat anti-rabbit HRP-conjugated antibody (Sangon) for 1 h at room temperature, washed with TBST, and subsequently imaged using ChemiDoc (Bio-Rad).

Particularly, the blocked membranes were directly probed with a rabbit anti-mouse HRP-conjugated antibody (Sangon) as the ZZ region could bind to the secondary antibody.

Co-immunoprecipitation

The supernatant of cell lysate of mamC-ZZ or purified mamC-ZZ was incubated with purified FLAG-tagged scFv-Fc overnight at 4°C. Afterwards, the mixture was incubated with anti-FLAG resin (GenScript, Nanjing, CN) for 1 h. The FLAG-tagged proteins scFv-Fc and the interacted mamC-ZZ from the lysate were then immobilized on the resin, whereas the unbound proteins were washed away with TBS. Subsequently, the protein–protein complex was eluted with acid elution buffer. The elution was then digested by thrombin to cut off GST region. The product was then analyzed by SDS–PAGE and western blotting. For negative control, only purified scFv-Fc was used as input.

Cell Culture

MDA-MB-453 and MDA-MB-231 cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere with 5% CO₂ at 37°C. All cells were passaged at 80% confluency.

Targeted Cell Uptake Evaluation of scFv-Fc Using Flow Cytometry

MDA-MB-453 and MDA-MB-231 cells at 80% confluence were collected by centrifugation after treatment using 0.5% trypsin-EDTA and resuspended in 50 μL PBS. Then, the cells were washed thrice with PBS, at 400 g for 5 min. Next, the cells were incubated (4°C in dark) by scFv-Fc (or goat anti-rabbit IgG H&L (HRP) as isotype control), rabbit anti-DDDDK tag (binds to FLAG^® tag sequence) antibody, goat anti-rabbit IgG H&L (Alexa Fluor^® 488) and Fixable Viability Dye eFluor^TM 450 (eBioscienc^TM), during each incubation the cells were washed thrice with PBS, centrifuge at 400 rcf for 5 mins. In the end, the cells were resuspended in 300 μL PBS, and the Alexa Fluor^® 488 and Fixable Viability Dye eFluo^TM 450 fluorescence of the cells was immediately analyzed using a flow cytometer (CytoFLEX, Beckman Coulter Inc., USA).

Culture of Magnetotactic Bacteria

Magnetotacitc bacteria is a general term refers to a group of species. Here, we choose Magnetospirillum gryphiswaldense strain MSR-1 (BCRC17316) as our target. MSR-1 was purchased from Beaver County Rehabilitation Center(Taiwan, China).

The culture medium(BCRC medium no.611) of M. Gryphiswaldense MSR-1 is prepared as follows.

Magntospirillum medium
KH₂PO₄	0.68g
Na-thioglycolate	0.05g
L(+)-Tartaric acid	0.37g
Succinic acid	0.37g
Trace elements solution	5ml
Resazurin	0.5mg
Distilled water	1000ml
NaNO₃	0.12g
Na-acetate	0.05g
Vitamin solution	10ml
Fe(III) quinate solution	Hyundai
Dissolve components in the order given. Adjust pH to 6.75 with NaOH and boil medium for 1 min. Purge medium with N₂ gas for 10 min. Under the same atmosphere, anaerobically fill tubes to 1/3 of their volume and seal. Autoclave at 121℃ for 15 min. Before inoculation, add.

Trace elements solution
Nitrilotriacetic acid	1.5g
MgSO₄·7H₂O	3g
MnSO₄·2H₂O	0.5g
NaCl	1g
CaCl₂·2H₂O	0.1g
ZnSO₄·7H₂O	0.18g
CuSO₄·5H₂O	0.01g
Kal(SO₄)2·12H₂O	0.02g
H₃3BO₃	0.05g
Na₂MoO₄·2H₂O	0.01g
Distilled water	1000mL
FeSO₄·7H₂O	0.1g
CoSO₄·7H₂O	0.18g
NiCl₂·6H₂O	0.025g
Na₂SeO₃·5H₂O	0.3mg
Na₂WO₄·2H₂O	0.4mg
Trace elements solution:First dissolve nitrilotriacetic acid adjust pH to 6.5 with KOH, then add minerals. Final pH 7.0 (with KOH).

Vitamin solution
Biotin	2mg
Folic acid	2mg
Pyridoxine-HCl	10mg
Thiamine-HCl·2H₂₀	5mg
Riboflavin	5mg
Vitamin B₁₂	0.1mg
p-Aminobenzoic acid	5mg
Lipoic acid	5mg
Distilled water	1000mL
Nicotinic acid	5mg
D-Ca-pantothenate	5mg
Dissolve components in the order given. Adjust pH to 6.75 with NaOH and boil medium for 1 min. Purge medium with N₂ gas for 10 min. Under the same atmosphere, anaerobically fill tubes to 1/3 of their volume and seal. Autoclave at 121℃ for 15 min. Before inoculation, add.

Ferric quinate solution, 0.01 M
FeCl₃·6H₂O	0.45g
Quinic acid	0.19g
Distilled water	100mL
Ferric quinate solution:Dissolve and autoclave at 121℃ for 15 min.

In the incubation period, aerobic environment is conducive to the growth of M.gryphiswaldense strain MSR-1.However, MicroAeroPack or AnaeroPack is needed for fermentation period. The MicroAeroPack or AnaeroPack was purchased from Mitsubishi Gas Chemical(Tokyo, Japan).

Bacterial PCR of MSR-1

Because magnetotactic bacteria grew too slowly to form a visible colony, we tent to demonstrate that it was truly grown in the medium. The liquid medium was centrifuged at 12000 rpm for 10 minutes and the sediment was used for bacterial PCR using specific primers designed from the 16srDNA of Magnetospirillum gryphiswaldense, E.coli and Agrobacterium were used in the bacterial PCR as the negative control.

References

[1]. Ahmadzadeh, M., Farshdari, F., Nematollahi, L., Behdani, M., & Mohit, E. (2020). Anti-HER2 scFv Expression in Escherichia coli SHuffle^®T7 Express Cells: Effects on Solubility and Biological Activity. Molecular biotechnology, 62(1), 18–30. https://doi.org/10.1007/s12033-019-00221-2

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+                             <li><a href="https://2020.igem.org/Team:ZJU-China/Model">Model</a></li>
-                             <li><a href="services-classic.html">xxx Model</a></li>
+                             <li><a href="https://2020.igem.org/Team:ZJU-China/Engineering">Engineering</a></li>
+                            <li><a href="https://2020.igem.org/Team:ZJU-China/Proof_Of_Concept">Proof of Concept</a></li>
                          </ul>
@@ Line 144: / Line 275: @@
                          <a class="nav-link" href="#">Parts</a>
                          <ul class="dropdown-list">
-                             <li><a href="about.html">Measurement</a></li>
+                             <li><a href="https://2020.igem.org/Team:ZJU-China/Contribution">Contribution</a></li>
-                             <li><a href="pricing-table.html">Improvement</a></li>
+                             <li><a href="https://2020.igem.org/Team:ZJU-China/Improve">Improve</a></li>
-                             <li><a href="login.html">New Parts</a></li>
+                             <li><a href="https://2020.igem.org/Team:ZJU-China/Newparts">New Parts</a></li>
                          </ul>
@@ Line 153: / Line 284: @@
                          <a class="nav-link" href="#">Human</a>
                          <ul class="dropdown-list">
-                             <li><a href="safety.html">Safety</a></li>
+                             <li><a href="https://2020.igem.org/Team:ZJU-China/Human_Practices">Human Practices</a></li>
-                             <li><a href="blog-classic.html">Human Practice</a></li>
+                             <li><a href="https://2020.igem.org/Team:ZJU-China/Education">Education</a></li>
+                            <li><a href="https://2020.igem.org/Team:ZJU-China/Collaborations">Collaborations</a></li>
+                            <li><a href="https://2020.igem.org/Team:ZJU-China/Partnership">Partnership</a></li>
+                            <li><a href="https://2020.igem.org/Team:ZJU-China/Safety">Safety</a></li>
-                        </ul>
+                            <li><a href="https://2020.igem.org/Team:ZJU-China/Implementation">Implementation</a></li>
-                    </li>
+                             <li><a href="https://2020.igem.org/Team:ZJU-China/Entrepreneurship">Entrepreneurship</a></li>
-                    <li class="nav-item dropdown-box" style="background-color:orchid;opacity: 0.9;">
-                        <a class="nav-link" href="#">Application</a>
-                        <ul class="dropdown-list">
-                            <li><a href="blog-grid.html">Hardware</a></li>
-                             <li><a href="blog-classic.html">Software</a></li>
                          </ul>
                      </li>
                  </ul>
@@ Line 175: / Line 307: @@
      <!-- Mobile Menu Start -->
      <nav class="mobile_menu hidden d-none">
-         <a href="index.html"><img class="mobile-logo" src="assets/img/teamt.png" alt="Logo"></a>
+         <a href="https://2020.igem.org/Team:ZJU-China"><img class="mobile-logo" src="https://static.igem.org/mediawiki/2020/7/7f/T--ZJU-China--temt.png" alt="Logo"></a>
          <ul class="nav navbar-nav navbar-right menu">
              <li class="dropdown active">
@@ Line 184: / Line 316: @@
                  <a>Team</a>
                  <ul class="sub_menu">
-                     <li><a href="team.html">Team Members</a></li>
+                     <li><a href="https://2020.igem.org/Team:ZJU-China/Team">Members</a></li>
-                     <li><a href="domain.html">Attributions</a></li>
+                     <li><a href="https://2020.igem.org/Team:ZJU-China/Attributions">Attributions</a></li>
+                    <li><a href="https://2020.igem.org/Team:ZJU-China/Medals">Medals</a></li>
                  </ul>
              </li>
@@ Line 191: / Line 325: @@
                  <a>Project</a>
                  <ul class="sub_menu">
-                     <li><a href="domain.html">Description</a></li>
+                     <li><a href="https://2020.igem.org/Team:ZJU-China/Description">Description</a></li>
-                    <li><a href="background.html">Background</a></li>
+                            <li><a href="https://2020.igem.org/Team:ZJU-China/Background">Background</a></li>
-                    <li><a href="domain.html">Design</a></li>
+                            <li><a href="https://2020.igem.org/Team:ZJU-China/Design">Design</a></li>
-                    <li><a href="domain.html">Experiment</a></li>
+                            <li><a href="https://2020.igem.org/Team:ZJU-China/Magnetosome">Magnetosome</a></li>
-                    <li><a href="domain.html">Result</a></li>
+                            <li><a href="https://2020.igem.org/Team:ZJU-China/Experiments">Experiments</a></li>
-                    <li><a href="domain.html">Demonstrate</a></li>
+                            <li><a href="https://2020.igem.org/Team:ZJU-China/Results">Results</a></li>
+                            <li><a href="https://2020.igem.org/Team:ZJU-China/Future">Future</a></li>
                  </ul>
              </li>
              <li class="dropdown">
-                 <a>Modeling</a>
+                 <a>Model</a>
                  <ul class="sub_menu">
-                    <li><a href="domain.html">Overview</a></li>
+                     <li><a href="https://2020.igem.org/Team:ZJU-China/Model">Model</a></li>
-                    <li><a href="domain.html">xxx Model</a></li>
+                            <li><a href="https://2020.igem.org/Team:ZJU-China/Engineering">Engineering</a></li>
+                            <li><a href="https://2020.igem.org/Team:ZJU-China/Proof_Of_Concept">Proof of Concept</a></li>
                  </ul>
              </li>
@@ Line 209: / Line 346: @@
                  <a>Parts</a>
                  <ul class="sub_menu">
-                     <li><a href="domain.html">Measurement</a></li>
+                     <li><a href="https://2020.igem.org/Team:ZJU-China/Contribution">Contribution</a></li>
-                     <li><a href="domain.html">Improvement</a></li>
+                     <li><a href="https://2020.igem.org/Team:ZJU-China/Improve">Improve</a></li>
-                     <li><a href="domain.html">New Parts</a></li>
+                     <li><a href="https://2020.igem.org/Team:ZJU-China/Newparts">New Parts</a></li>
                  </ul>
              </li>
@@ Line 217: / Line 355: @@
                  <a>Human</a>
                  <ul class="sub_menu">
-                     <li><a href="safety.html">Safety</a></li>
+                     <li><a href="https://2020.igem.org/Team:ZJU-China/Human_Practices">Human Practices</a></li>
-                     <li><a href="blog-classic.html">Human Practice</a></li>
+                     <li><a href="https://2020.igem.org/Team:ZJU-China/Education">Education</a></li>
-                </ul>
+                    <li><a href="https://2020.igem.org/Team:ZJU-China/Collaborations">Collaborations</a></li>
-            </li>
+                    <li><a href="https://2020.igem.org/Team:ZJU-China/Partnership">Partnership</a></li>
-            <li class="dropdown">
+                    <li><a href="https://2020.igem.org/Team:ZJU-China/Safety">Safety</a></li>
-                <a>Application</a>
-                <ul class="sub_menu">
+                     <li><a href="https://2020.igem.org/Team:ZJU-China/Implementation">Implementation</a></li>
-                     <li><a href="blog-grid.html">Hardware</a></li>
+                     <li><a href="https://2020.igem.org/Team:ZJU-China/Entrepreneurship">Entrepreneurship</a></li>
-                     <li><a href="blog-classic.html">Software</a></li>
                  </ul>
              </li>
          </ul>
@@ Line 235: / Line 374: @@
 <!--Main Menu/ Mobile Menu Section-->
-<div class="pagename">
+<div id="swrapper">
-    <h1>Experiment</h1>
+<div id="mcontent" >
-</div>
+    <div class="pagename">
+        <h1>Experiments</h1>
+    </div>
-<div id="scrollable">
+     <div id="scrollable">
-         <div class="pagestyle" style="background-image: url('https://static.igem.org/mediawiki/2020/c/cd/T--ZJU-China--wiki_navback.jpg');top:120px;">
-             <div  class="section Cloning" id="Cloning">
+         <div class="pagestyle"
+            style="background-image: url('https://static.igem.org/mediawiki/2020/c/cd/T--ZJU-China--wiki_navback.jpg');">
+             <div class="section Cloning" id="Cloning">
                  <div class="container1">
-                <h2>Cloning</h2>
+                    <h2>Cloning</h2>
-                <p>
-                    For cloning of the fusion of single-chain variable fragment (scFv) and fragment crystallizable (Fc),
-                    the amino acid sequence of anti-HER2 scFv described by Ahmadzadeh, M. <a href="#our-team"><sup>[1]</sup></a>and the CH2 & CH3 region of immunoglobulin heavy constant gamma 1,
-                    which is the constant region of immunoglobulin heavy chains <a href="https://www.uniprot.org/uniprot/P01857" target="_blank">(UniProtKB-P01857)</a> ,
-                    was used respectively. In addition, the hinge region of immunoglobulin heavy constant gamma 1 was used as the linker between scFv and Fc,
-                    a FLAG tag was fused to the C-terminus as well. Furthermore, we employed codon optimization to maximize the expression of a functional protein in <i>Escherichia coli</i>.
-                    The gene was synthesized by GenScript Biotech Corporation (Nanjing, CN). The gene was subcloned into gene expression vector pET19b, introduced a 10× histidine tag at the N-terminus.
-                </p>
-                <br>
-                <br>
-                <div class="imgbox">
-                <img src="https://static.igem.org/mediawiki/2020/5/56/T--ZJU-China--wiki_experiment_fig1.png"></img>
-                <h6>Fig1.Vector of scFv-Fc, pET19b.</h6>
-                </div>
-                <br>
-                <p>For cloning of the fusion of mamC <a href="https://www.ncbi.nlm.nih.gov/protein/AVM72836.1" target="_blank">(GeneBank: AVM72836.1)</a>
-                    and ZZ <a href="https://www.ncbi.nlm.nih.gov/nuccore/M74186.1" target="_blank">(GeneBank:M74186.1 )</a> , the gene was synthesized and
-                    codon optimized by GenScript Biotech Corporation (Nanjing, CN). The gene was subcloned into gene expression vector pGEX-2TK,
-                    introduced a GST tag at the N-terminus.</p>
-                <div class="imgbox">
-                    <img src="https://static.igem.org/mediawiki/2020/8/87/T--ZJU-China--wiki_experiment_fig2.png"></img>
-                    <h6>Fig2.Vector of mamC_zz, pGEX-2TK.</h6>
-                </div>
-                <br>
-                <br>
-                <h2>Expression of scFv-Fc</h2>
+                    <p>
-                <p>
+                        For cloning of the fusion of <b>single-chain variable fragment (scFv)</b> and <b>fragment crystallizable
-                    After the DNA sequence was confirmed (SUNYA, Zhejiang, CN), the pET19b was transformed into
+                        (Fc)</b>,
-                    <i>Escherichia coli</i> SHuffle<sup>®</sup>T7 Express Cells. The cells were grown in LB medium at 37°C until the
+                        the amino acid sequence of anti-HER2 scFv described by Ahmadzadeh, M.<a
-                    OD600 reached 0.6, at which time protein expression was induced by adding filter sterilized
+                            href="#references"><sup>[1]</sup></a> and the CH2 & CH3 region of immunoglobulin heavy constant
-                    isopropyl-β-D-thiogalactopyranoside (IPTG). The cells were then incubated at 30°C for an additional
+                        gamma 1,
-                    time to expression. Afterwards, the cells were harvested by centrifugation at 4000 rpm for 12 min.
+                        which is the constant region of immunoglobulin heavy chains <a
-                    The cell pellet was resuspended in lysis buffer (50mM Tris/HCl, pH 7.5, 500mM NaCl, 0.1% CA630, 10%
+                            href="https://www.uniprot.org/uniprot/P01857" target="_blank" class="outerlink" style="color:#0a8aff;">(UniProtKB-P01857)</a>,
-                    glycerol and 5mM β-mercaptoethanol) and the cells were lysed by sonication. The cell extraction was
+                        was used respectively. In addition, the hinge region of immunoglobulin heavy constant gamma 1
-                    clarified by centrifugation at 10000 rpm for 15 min.
+                        was used as the linker between scFv and Fc,
-                </p>
+                        a FLAG tag was fused to the C-terminus as well. Furthermore, we employed codon optimization to
+                        maximize the expression of a functional protein in <i>Escherichia coli</i>.
-                <h2>Expression of mamC-ZZ</h2>
+                        The gene was synthesized by GenScript Biotech Corporation (Nanjing, CN). The gene was subcloned
-                <p>
+                        into gene expression vector pET19b, introduced a 10× histidine tag at the N-terminus.
-                    After the DNA sequence was confirmed (SUNYA, Zhejiang, CN), the pGEX-2TK construct was transformed into
+                    </p>
-                    BL21 (DE3). The process of growth, induction and harvest was the same as that of scFv-Fc. The cell pellet
+                    <br>
-                    was resuspended in lysis buffer (50mM Tris-HCl pH8.0 5mM EDTA and 1.5mM NaCl, phenylmethylsulfonyl fluoride).
+                    <br>
-                    Add lysozyme (Solarbio, Beijing, CN) to a final concentration of 500 μg/mL, incubating for 30 min at room
+                    <div class="imgbox">
-                    temperature and centrifuging at 12000 rpm for 30 seconds. Discard the supernatant to remove the periplasm
+                        <img src="https://static.igem.org/mediawiki/2020/5/56/T--ZJU-China--wiki_experiment_fig1.png" style="max-width: 70%;"></img>
-                    protein. Add RIPA IV (Sangon, Shanghai, CN) and incubate on ice for 10 min, vibrating the tubes for several
+                        <h6>Figure 1. Vector of scFv-Fc, pET19b.</h6>
-                    times during the incubation. The cell extract was clarified by centrifugation at 10000 rpm for 10 min.
+                    </div>
-                </p>
+                    <br>
-                <h2>Purification of scFv-Fc</h2>
+                    <p>For cloning of the fusion of mamC <a class="outerlink" style="color:#0a8aff;" href="https://www.ncbi.nlm.nih.gov/protein/AVM72836.1" target="_blank">(GeneBank: AVM72836.1)</a>
-                <p>
+                        and ZZ <a class="outerlink" style="color:#0a8aff;"  href="https://www.ncbi.nlm.nih.gov/nuccore/M74186.1"
-                    The protein was purified by immunoprecipitation (IP) using anti‐DYKDDDDK G1 affinity
+                            target="_blank">(GeneBank:M74186.1 )</a>, the gene was synthesized and
-                    resin (GenScript, Nanjing, CN). Resuspend the resin to form a uniform slurry and transfer
+                        codon optimized by GenScript Biotech Corporation (Nanjing, CN). The gene was subcloned into gene
-μL of the slurry into a 1.5 mL vial. Add 500 μL TBS into the vial and gently mix the
+                        expression vector pGEX-2TK,
-                    resin, centrifuge at 6000 rcf for 30 seconds, and remove supernatant carefully. Repeat
+                        introduced a GST tag at the N-terminus.</p>
-                    this step for three times. Add 1 mL supernatant of the cell lysate to the washed resin.
-                    Mix by end‐to‐end rotation on a tube rotator overnight at 4°C. To remove the nonspecific
+                    <div class="imgbox">
-                    binding segment, centrifuge at 6000 rcf for 30 seconds, carefully remove supernatant and add
+                        <img src="https://static.igem.org/mediawiki/2020/8/87/T--ZJU-China--wiki_experiment_fig2.png" style="max-width: 70%;"></img>
-μL TBS to wash the resin three times. Remove as much supernatant as possible without
+                        <h6>Figure 2. Vector of mamC_ZZ, pGEX-2TK.</h6>
-                    disturbing the resin, add 120 μL of acid elution buffer (0.1 M glycine HCl, pH 3.5) into
-                    the washed resin and use a wide bore pipette tip to gently resuspend the resin. Incubate
+                    </div>
-                    at room temperature for 5 minutes, mix gently by tapping the tube once or twice during the
-                    incubation period. After incubation, centrifuge at 6000 rcf for 30 seconds. Carefully transfer
+                    <br>
-                    supernatant into a new vial containing 5 μL 1 M Tris, pH 9.0 for further application.
+                    <br>
-                </p>
+                    <h2>Expression of scFv-Fc</h2>
+                    <p>
+                        After the DNA sequence was confirmed (SUNYA, Zhejiang, CN), the pET19b was transformed into
+                        <i>Escherichia coli</i> SHuffle<sup>®</sup>T7 Express Cells. The cells were grown in LB medium
+                        at 37°C until the
+                        OD600 reached 0.6, at which time protein expression was induced by adding filter sterilized
+                        isopropyl-β-D-thiogalactopyranoside (IPTG). The cells were then incubated at 30°C for an
+                        additional
+                        time to expression. Afterwards, the cells were harvested by centrifugation at 4000 rpm for 12
+                        min.
+                        The cell pellet was resuspended in lysis buffer (50 mM Tris/HCl, pH 7.5, 500 mM NaCl, 0.1% CA630,
+%
+                        glycerol and 5 mM β-mercaptoethanol) and the cells were lysed by sonication. The cell extraction
+                        was
+                        clarified by centrifugation at 10000 rpm for 15 min.
+                    </p>
+                    <h2>Expression of mamC-ZZ</h2>
+                    <p>
+                        After the DNA sequence was confirmed (SUNYA, Zhejiang, CN), the pGEX-2TK construct was
+                        transformed into
+                        BL21 (DE3). The process of growth, induction and harvest was the same as that of scFv-Fc. The
+                        cell pellet team
+                        was resuspended in lysis buffer (50 mM Tris-HCl pH8.0 5 mM EDTA and 1.5 mM NaCl,
+                        phenylmethylsulfonyl fluoride).
+                        Add lysozyme (Solarbio, Beijing, CN) to a final concentration of 500 μg/mL, incubating for 30
+                        min at room
+                        temperature and centrifuging at 12000 rpm for 30 seconds. Discard the supernatant to remove the
+                        periplasm
+                        protein. Add RIPA IV (Sangon, Shanghai, CN) and incubate on ice for 10 min, vibrating the tubes
+                        for several
+                        times during the incubation. The cell extract was clarified by centrifugation at 10000 rpm for
+min.
+                    </p>
+                    <h2>Purification of scFv-Fc</h2>
+                    <p>
+                        The protein was purified by immunoprecipitation (IP) using anti‐DYKDDDDK G1 affinity
+                        resin (GenScript, Nanjing, CN). Resuspend the resin to form a uniform slurry and transfer
+μL of the slurry into a 1.5 mL vial. Add 500 μL TBS into the vial and gently mix the
+                        resin, centrifuge at 6000 rcf for 30 seconds, and remove supernatant carefully. Repeat
+                        this step for three times. Add 1 mL supernatant of the cell lysate to the washed resin.
+                        Mix by end‐to‐end rotation on a tube rotator overnight at 4°C. To remove the nonspecific
+                        binding segment, centrifuge at 6000 rcf for 30 seconds, carefully remove supernatant and add
+μL TBS to wash the resin three times. Remove as much supernatant as possible without
+                        disturbing the resin, add 120 μL of acid elution buffer (0.1 M glycine HCl, pH 3.5) into
+                        the washed resin and use a wide bore pipette tip to gently resuspend the resin. Incubate
+                        at room temperature for 5 minutes, mix gently by tapping the tube once or twice during the
+                        incubation period. After incubation, centrifuge at 6000 rcf for 30 seconds. Carefully transfer
+                        supernatant into a new vial containing 5 μL 1 M Tris, pH 9.0 for further application.
+                    </p>
                  </div>
              </div>
-             <div  class="section about" id="about">
+             <div class="section about" id="about">
                  <div class="container1">
-                        <h2>Optimum concentration of IPTG for expressing</h2>
-                        <p>
-                            The transformed cells were grown until the OD<sub>600</sub> reached 0.6, at which time
-                            protein expression was induced by a gradient from 0 to 2mM of IPTG. The cells
-                            were then incubated at 30°C for an additional 24 h.
-                        </p>
-                        <br>
+                    <h2>Optimum Concentration of IPTG for Expressing</h2>
-                         <br>
+                    <p>
+                         The transformed cells were grown until the OD600 reached 0.6, at which time
+                        protein expression was induced by a gradient from 0 to 2 mM of IPTG. The cells
+                        were then incubated at 30°C for an additional 24 h.
+                    </p>
-                        <h2>Optimum induction time for expressing</h2>
+                    <br>
+                    <br>
-                        <p>
-                            The transformed cells were grown until the OD<sub>600</sub> reached 0.6, at which time
+                    <h2>Optimum Induction Time for Expressing</h2>
-                            protein expression was induced by 2mM IPTG. The cells were then incubated at
-°C for a gradient from 0 to 24 h. For negative control, none inducer was
+                    <p>
-                            introduced.
+                        The transformed cells were grown until the OD<sub>600</sub> reached 0.6, at which time
-                        </p>
+                        protein expression was induced by 2 mM IPTG. The cells were then incubated at
+°C for a gradient from 0 to 24 h. For negative control, none inducer was
+                        introduced.
-                 </div>
+                    </p>
+                 </div>
              </div>
-             <div  class="section services" id="services">
+             <div class="section services" id="services">
                  <div class="container1">
-                     <h2 style="line-height:1.5;">Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)</h2>
+                     <h2 style="line-height:1.5;">Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS–PAGE)
+                    </h2>
                      <br>
                      <br>
                      <p>
-                         The cell lysate or purified protein was mixed 1:1 (v:v) with 2× loading buffer
+                         The cell lysate or purified protein was mixed 1:1 (v:v) with 2× loading buffer (TIANGEN, Beijing, CN), heated for 5 min at 95°C and subjected to SDS–PAGE (5%
-                        (TIANGEN,Beijing,CN), heated for 5 min at 95°C and subjected to SDS–PAGE (5%
+                         stacking gel, 8% separating gel). Gels were run at 110V for 15vmin and then at 160V
-                         stacking gel, 8% separating gel). Gels were run at 110V for 15min and then at 160V
                          for ~50 min in running buffer.
                      </p>
-                     <h2>Coomassie staining</h2>
+                     <h2>Coomassie Staining</h2>
                      <p>
                          Following SDS–PAGE, gels was stained with Coomassie brilliant blue (0.1% Coomassie
                          brilliant blue R-250, 25% isopropanol, 10% acetic acid) for 2 h at room temperature.
                          Destain in destaining buffer (5% ethanol, 10% acetic acid) overnight.
                      </p>
-                     <h2>Western-blotting of proteins</h2>
+                     <h2>Western Blotting of Proteins</h2>
                      <p>
                          Following SDS–PAGE, samples were transferred to nitrocellulose membrane (Merck) by
                          semi-dry transfer (Bio-Rad). Membranes were blocked 1 h at room temperature in 5%
                          (w/v) nonfat milk in TBST, probed with a rabbit anti-FLAG antibody (Abcam) overnight
                          at 4°C, washed with TBST, probed with a goat anti-rabbit HRP-conjugated antibody
                          (Sangon) for 1 h at room temperature, washed with TBST, and subsequently imaged using
-                        ChemiDoc (Bio-Rad).Particularly, the blocked membranes were directly probed with a rabbit
+						ChemiDoc (Bio-Rad).
-                         anti-mouse HRP-conjugated antibody (Sangon) as the ZZ region could bind to the secondary antibody.
+					</p>
+					<br>
+					<p>
+						Particularly, the blocked membranes were directly probed with a rabbit
+                         anti-mouse HRP-conjugated antibody (Sangon) as the ZZ region could bind to the secondary
+                        antibody.
                      </p>
                      <h2>Co-immunoprecipitation</h2>
                      <p>
                          The supernatant of cell lysate of mamC-ZZ or purified mamC-ZZ was incubated with
                          purified FLAG-tagged scFv-Fc overnight at 4°C. Afterwards, the mixture was incubated
                          with anti-FLAG resin (GenScript, Nanjing, CN) for 1 h. The FLAG-tagged proteins scFv-Fc
                          and the interacted mamC-ZZ from the lysate were then immobilized on the resin, whereas
                          the unbound proteins were washed away with TBS. Subsequently, the protein–protein complex
                          was eluted with acid elution buffer. The elution was then digested by thrombin to cut off
-                         GST region. The product was then analyzed by SDS-PAGE and western blotting. For negative
+                         GST region. The product was then analyzed by SDS–PAGE and western blotting. For negative
                          control, only purified scFv-Fc was used as input.
                      </p>
                  </div>
              </div>
              <div class="section showcase" id="showcase">
                  <div class="container1">
-                     <h2>Cell culture</h2>
+                     <h2>Cell Culture</h2>
                      <p>
-                         MDA-MB-453, MDA-MB-231, MCF-7, and MDA-MB-468 cells were cultured in
+                         MDA-MB-453 and MDA-MB-231 cells were cultured in high glucose Dulbecco’s modified Eagle’s medium
-                        high glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented
+                        (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere with 5%
-                        with 10% fetal bovine serum (FBS, Gibco) in a humidified atmosphere with
+                         CO<sub>2</sub> at 37°C. All cells were passaged at 80% confluency.</p>
-% CO2 at 37°C. All cells were passaged at 80% confluency.</p>
+                    <br><br><br>
-                        <br><br><br>
+                     <h2 style="line-height:1.5;">Targeted Cell Uptake Evaluation of scFv-Fc Using Flow Cytometry</h2>
-                     <h2 style="line-height:1.5;">Targeted cell uptake evaluation of scFv-Fc using flow cytometry</h2>
                      <br>
                      <br>
                      <p>
-                         MDA-MB-453, MDA-MB-231, MCF-7, and MDA-MB-468 cells at 90% confluence were
+                         MDA-MB-453 and MDA-MB-231 cells at 80% confluence were collected by centrifugation after
-                         collected, respectively, and seeded in a six-well plate. After overnight incubation,
+                         treatment using 0.5% trypsin-EDTA and resuspended in 50 μL PBS. Then, the cells were washed
-                        the media were replaced with the fresh media containing 30 μg/mL of scFv-Fc and further
+                        thrice with PBS, at 400 g for 5 min. Next, the cells were incubated (4°C in dark) by scFv-Fc (or
-                         incubated for 1, 3, and 6 h. The cells were then collected by centrifugation after treatment
+                         goat anti-rabbit IgG H&L (HRP) as isotype control), rabbit anti-DDDDK tag (binds to
-                         using 0.5% trypsin-EDTA and washed thrice with PBS. In the end, the cells were resuspended in
+                         FLAG<sup>®</sup> tag sequence) antibody, goat anti-rabbit IgG H&L (Alexa Fluor<sup>®</sup> 488)
-μL PBS, and the Alexa Fluor<sup>®</sup> 488 fluorescence of the cells was immediately analyzed using
+                        and Fixable Viability Dye eFluor<sup>TM</sup> 450 (eBioscienc<sup>TM</sup>), during each
-                        a flow cytometer (CytoFLEX, Beckman Coulter Inc., USA).
+                        incubation the cells were washed thrice with PBS, centrifuge at 400 rcf for 5 mins. In the end, the cells were
+                         resuspended in 300 μL PBS, and the Alexa Fluor<sup>®</sup> 488 and Fixable Viability Dye
+                        eFluo<sup>TM</sup> 450 fluorescence of the cells was immediately analyzed using a flow cytometer
+                        (CytoFLEX, Beckman Coulter Inc., USA).
                      </p>
-                     <h2>Culture of Magnetotactic bacteria</h2>
+                     <h2>Culture of Magnetotactic Bacteria</h2>
                      <p>
-                         Magnetotacitc bacteria is a general term refers to a group of species. Here, we choose <i>Magnetospirillum gryphiswaldense</i>
+                         Magnetotacitc bacteria is a general term refers to a group of species. Here, we choose
-                         strain MSR-1 (BCRC17316) as our target. MSR-1 was purchased from Beaver County Rehabilitation
+                        <i>Magnetospirillum gryphiswaldense</i>
+                         strain MSR-1 (BCRC17316) as our target. MSR-1 was purchased from Beaver County Rehabilitation
                          Center(Taiwan, China).
                      </p>
@@ Line 421: / Line 593: @@
                      <div class="imgbox">
-                    <table class="pure-table pure-table-bordered">
+                        <table class="pure-table pure-table-bordered">
-                        <thead>
+                            <thead>
-                            <tr>
+                                <tr>
-                                <th colspan="2">Magntospirillum medium</th>
+                                    <th colspan="2">Magntospirillum medium</th>
-                            </tr>
+                                </tr>
-                        </thead>
+                            </thead>
-                        <tbody>
+                            <tbody>
-                            <tr>
+                                <tr>
-                                <td>KH<sub>2</sub>PO<sub>4</sub></td>
+                                    <td>KH<sub>2</sub>PO<sub>4</sub></td>
-                                <td>0.68g</td>
+                                    <td>0.68g</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Na-thioglycolate</td>
+                                    <td>Na-thioglycolate</td>
-                                <td>0.05g</td>
+                                    <td>0.05g</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>L(+)-Tartaric acid</td>
+                                    <td>L(+)-Tartaric acid</td>
-                                <td>0.37g</td>
+                                    <td>0.37g</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Succinic acid</td>
+                                    <td>Succinic acid</td>
-                                <td>0.37g</td>
+                                    <td>0.37g</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Trace elements solution</td>
+                                    <td>Trace elements solution</td>
-                                <td>5ml</td>
+                                    <td>5ml</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Resazurin</td>
+                                    <td>Resazurin</td>
-                                <td>0.5mg</td>
+                                    <td>0.5mg</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Distilled water</td>
+                                    <td>Distilled water</td>
-                                <td>1000ml</td>
+                                    <td>1000ml</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>NaNO<sub>3</sub></td>
+                                    <td>NaNO<sub>3</sub></td>
-                                <td>0.12g</td>
+                                    <td>0.12g</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Na-acetate</td>
+                                    <td>Na-acetate</td>
-                                <td>0.05g</td>
+                                    <td>0.05g</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Vitamin solution</td>
+                                    <td>Vitamin solution</td>
-                                <td>10ml</td>
+                                    <td>10ml</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Fe(III) quinate solution</td>
+                                    <td>Fe(III) quinate solution</td>
-                                <td>Hyundai</td>
+                                    <td>Hyundai</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td colspan="2">
+                                    <td colspan="2">
-                                Dissolve components in the order given. Adjust pH to 6.75 with NaOH and
+                                        Dissolve components in the order given. Adjust pH to 6.75 with NaOH and
-                                boil medium for 1 min. Purge medium with N<sub>2</sub> gas for 10 min. Under the same
+                                        boil medium for 1 min. Purge medium with N<sub>2</sub> gas for 10 min. Under the
-                                atmosphere, anaerobically fill tubes to 1/3 of their volume and seal. Autoclave
+                                        same
-                                at 121℃ for 15 min. Before inoculation, add.
+                                        atmosphere, anaerobically fill tubes to 1/3 of their volume and seal. Autoclave
-                                </td>
+                                        at 121℃ for 15 min. Before inoculation, add.
-                            </tr>
+                                    </td>
-                        </tbody>
+                                </tr>
-                    </table>
+                            </tbody>
+                        </table>
                      </div>
                      <div class="imgbox">
-                    <table class="pure-table pure-table-bordered" style="margin-left:0;margin-right:0;">
+                        <table class="pure-table pure-table-bordered" style="margin-left:0;margin-right:0;">
-                        <thead>
+                            <thead>
-                            <tr>
+                                <tr>
-                                <th colspan="2">Trace elements solution</th>
+                                    <th colspan="2">Trace elements solution</th>
-                            </tr>
+                                 </tr>
-                        </thead>
+                             </thead>
-                        <tbody>
-                            <tr>
-                                 <td>Nitrilotriacetic acid</td>
-                                <td>1.5g</td>
-                            </tr>
-                             <tr>
-                                <td>MgSO<sub>4</sub>·7H<sub>2</sub>O</td>
-                                <td>3g</td>
-                            </tr>
-                            <tr>
-                                <td>MnSO<sub>4</sub>·2H<sub>2</sub>O</td>
-                                <td>0.5g</td>
-                            </tr>
-                            <tr>
-                                <td>NaCl</td>
-                                <td>1g</td>
-                            </tr>
-                            <tr>
-                                <td>CaCl<sub>2</sub>·2H<sub>2</sub>O</td>
-                                <td>0.1g</td>
-                            </tr>
-                            <tr>
-                                <td>ZnSO<sub>4</sub>·7H<sub>2</sub>O</td>
-                                <td>0.18g</td>
-                            </tr>
-                            <tr>
-                                <td>CuSO<sub>4</sub>·5H<sub>2</sub>O</td>
-                                <td>0.01g</td>
-                            </tr>
-                            <tr>
-                                <td>Kal(SO<sub>4</sub>)2·12H<sub>2</sub>O</td>
-                                <td>0.02g</td>
-                            </tr>
-                            <tr>
-                                <td>H<sub>3</sub>3BO<sub>3</sub></td>
-                                <td>0.05g</td>
-                            </tr>
-                            <tr>
-                                <td>Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O</td>
-                                <td>0.01g</td>
-                            </tr>
-                            <tr>
-                                <td>Distilled water</td>
-                                <td>1000mL</td>
-                            </tr>
-                             <tr>
+                             <tbody>
-                                 <td>FeSO<sub>4</sub>·7H<sub>2</sub>O</td>
+                                <tr>
-                                 <td>0.1g</td>
+                                    <td>Nitrilotriacetic acid</td>
-                            </tr>
+                                    <td>1.5g</td>
-                            <tr>
+                                 </tr>
-                                 <td>CoSO<sub>4</sub>·7H<sub>2</sub>O</td>
-                                <td>0.18g</td>
+                                <tr>
-                            </tr>
+                                    <td>MgSO<sub>4</sub>·7H<sub>2</sub>O</td>
-                            <tr>
+                                    <td>3g</td>
-                                 <td>NiCl<sub>2</sub>·6H<sub>2</sub>O</td>
+                                 </tr>
-                                <td>0.025g</td>
-                            </tr>
+                                <tr>
-                            <tr>
+                                    <td>MnSO<sub>4</sub>·2H<sub>2</sub>O</td>
-                                <td>Na<sub>2</sub>SeO<sub>3</sub>·5H<sub>2</sub>O</td>
+                                    <td>0.5g</td>
-                                <td>0.3mg</td>
+                                </tr>
-                            </tr>
+                                <tr>
-                            <tr>
+                                    <td>NaCl</td>
-                                <td>Na<sub>2</sub>WO<sub>4</sub>·2H<sub>2</sub>O</td>
+                                    <td>1g</td>
-                                <td>0.4mg</td>
+                                </tr>
-                            </tr>
+                                <tr>
-                            <tr>
+                                    <td>CaCl<sub>2</sub>·2H<sub>2</sub>O</td>
-                                <td colspan="2">
+                                    <td>0.1g</td>
-                                    Trace elements solution:First dissolve nitrilotriacetic acid adjust pH to
+                                 </tr>
-.5 with KOH, then add minerals. Final pH 7.0 (with KOH).
+                                <tr>
-                                </td>
+                                    <td>ZnSO<sub>4</sub>·7H<sub>2</sub>O</td>
-                            </tr>
+                                    <td>0.18g</td>
-                        </tbody>
+                                </tr>
-                    </table>
+                                <tr>
+                                    <td>CuSO<sub>4</sub>·5H<sub>2</sub>O</td>
+                                    <td>0.01g</td>
+                                 </tr>
+                                <tr>
+                                    <td>Kal(SO<sub>4</sub>)2·12H<sub>2</sub>O</td>
+                                    <td>0.02g</td>
+                                </tr>
+                                <tr>
+                                    <td>H<sub>3</sub>3BO<sub>3</sub></td>
+                                    <td>0.05g</td>
+                                </tr>
+                                <tr>
+                                    <td>Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O</td>
+                                    <td>0.01g</td>
+                                </tr>
+                                <tr>
+                                    <td>Distilled water</td>
+                                    <td>1000mL</td>
+                                </tr>
+                                <tr>
+                                    <td>FeSO<sub>4</sub>·7H<sub>2</sub>O</td>
+                                    <td>0.1g</td>
+                                </tr>
+                                <tr>
+                                    <td>CoSO<sub>4</sub>·7H<sub>2</sub>O</td>
+                                    <td>0.18g</td>
+                                </tr>
+                                <tr>
+                                    <td>NiCl<sub>2</sub>·6H<sub>2</sub>O</td>
+                                    <td>0.025g</td>
+                                </tr>
+                                <tr>
+                                    <td>Na<sub>2</sub>SeO<sub>3</sub>·5H<sub>2</sub>O</td>
+                                    <td>0.3mg</td>
+                                </tr>
+                                <tr>
+                                    <td>Na<sub>2</sub>WO<sub>4</sub>·2H<sub>2</sub>O</td>
+                                    <td>0.4mg</td>
+                                </tr>
+                                <tr>
+                                    <td colspan="2">
+                                        Trace elements solution:First dissolve nitrilotriacetic acid adjust pH to
+.5 with KOH, then add minerals. Final pH 7.0 (with KOH).
+                                    </td>
+                                </tr>
+                            </tbody>
+                        </table>
                      </div>
                      <div class="imgbox">
-                    <table class="pure-table pure-table-bordered" style="margin-left:0;margin-right:0;">
+                        <table class="pure-table pure-table-bordered" style="margin-left:0;margin-right:0;">
-                        <thead>
+                            <thead>
-                            <tr>
+                                <tr>
-                                <th colspan="2">Vitamin solution</th>
+                                    <th colspan="2">Vitamin solution</th>
-                            </tr>
+                                </tr>
-                        </thead>
+                            </thead>
-                        <tbody>
+                            <tbody>
-                            <tr>
+                                <tr>
-                                <td>Biotin</td>
+                                    <td>Biotin</td>
-                                <td>2mg</td>
+                                    <td>2mg</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Folic acid</td>
+                                    <td>Folic acid</td>
-                                <td>2mg</td>
+                                    <td>2mg</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Pyridoxine-HCl</td>
+                                    <td>Pyridoxine-HCl</td>
-                                <td>10mg</td>
+                                    <td>10mg</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Thiamine-HCl·2H<sub>20</sub></td>
+                                    <td>Thiamine-HCl·2H<sub>20</sub></td>
-                                <td>5mg</td>
+                                    <td>5mg</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Riboflavin</td>
+                                    <td>Riboflavin</td>
-                                <td>5mg</td>
+                                    <td>5mg</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Vitamin B<sub>12</sub></td>
+                                    <td>Vitamin B<sub>12</sub></td>
-                                <td>0.1mg</td>
+                                    <td>0.1mg</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>p-Aminobenzoic acid</td>
+                                    <td>p-Aminobenzoic acid</td>
-                                <td>5mg</td>
+                                    <td>5mg</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Lipoic acid</td>
+                                    <td>Lipoic acid</td>
-                                <td>5mg</td>
+                                    <td>5mg</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Distilled water</td>
+                                    <td>Distilled water</td>
-                                <td>1000mL</td>
+                                    <td>1000mL</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>Nicotinic acid</td>
+                                    <td>Nicotinic acid</td>
-                                <td>5mg</td>
+                                    <td>5mg</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td>D-Ca-pantothenate</td>
+                                    <td>D-Ca-pantothenate</td>
-                                <td>5mg</td>
+                                    <td>5mg</td>
-                            </tr>
+                                </tr>
-                            <tr>
+                                <tr>
-                                <td colspan="2">
+                                    <td colspan="2">
-                                Dissolve components in the order given. Adjust pH to 6.75 with NaOH and
+                                        Dissolve components in the order given. Adjust pH to 6.75 with NaOH and
-                                boil medium for 1 min. Purge medium with N<sub>2</sub> gas for 10 min. Under the same
+                                        boil medium for 1 min. Purge medium with N<sub>2</sub> gas for 10 min. Under the
-                                atmosphere, anaerobically fill tubes to 1/3 of their volume and seal. Autoclave
+                                        same
-                                at 121℃ for 15 min. Before inoculation, add.
+                                        atmosphere, anaerobically fill tubes to 1/3 of their volume and seal. Autoclave
-                                </td>
+                                        at 121℃ for 15 min. Before inoculation, add.
-                            </tr>
+                                    </td>
-                        </tbody>
+                                </tr>
-                    </table>
+                            </tbody>
+                        </table>
                      </div>
                      <div class="imgbox">
-                    <table style="width: auto;height: auto;max-width: 100%;max-height: 100%;margin-right:auto ;margin-left:auto;" class="pure-table pure-table-bordered">
+                        <table
-                        <thead>
+                            style="width: auto;height: auto;max-width: 100%;max-height: 100%;margin-right:auto ;margin-left:auto;"
-                            <tr>
+                            class="pure-table pure-table-bordered">
-                                <th colspan="2">Ferric quinate solution, 0.01 M</th>
+                            <thead>
-                            </tr>
+                                <tr>
-                        </thead>
+                                    <th colspan="2">Ferric quinate solution, 0.01 M</th>
+                                </tr>
-                        <tbody>
+                            </thead>
-                            <tr>
-                                <td>FeCl<sub>3</sub>·6H<sub>2</sub>O</td>
+                            <tbody>
-                                <td>0.45g</td>
+                                <tr>
-                            </tr>
+                                    <td>FeCl<sub>3</sub>·6H<sub>2</sub>O</td>
+                                    <td>0.45g</td>
-                            <tr>
+                                </tr>
-                                <td>Quinic acid</td>
-                                <td>0.19g</td>
+                                <tr>
-                            </tr>
+                                    <td>Quinic acid</td>
+                                    <td>0.19g</td>
-                            <tr>
+                                </tr>
-                                <td>Distilled water</td>
-                                <td>100mL</td>
+                                <tr>
-                            </tr>
+                                    <td>Distilled water</td>
+                                    <td>100mL</td>
-                            <tr>
+                                </tr>
-                                <td colspan="2">
-                                    Ferric quinate solution:Dissolve and autoclave at 121℃ for
+                                <tr>
-min.
+                                    <td colspan="2">
-                                </td>
+                                        Ferric quinate solution:Dissolve and autoclave at 121℃ for
-                            </tr>
+min.
-                        </tbody>
+                                    </td>
-                    </table>
+                                </tr>
+                            </tbody>
+                        </table>
                      </div>
                      <br>
@@ Line 679: / Line 855: @@
                      <p>
-                         In the incubation period, aerobic environment is conducive to the growth of <i>M.gryphiswaldense</i> strain
+                         In the incubation period, aerobic environment is conducive to the growth of
-                         MSR-1.However, MicroAeroPack or AnaeroPack is needed for fermentation period. The MicroAeroPack or
+                        <i>M.gryphiswaldense</i> strain
+                         MSR-1.However, MicroAeroPack or AnaeroPack is needed for fermentation period. The MicroAeroPack
+                        or
                          AnaeroPack was purchased from Mitsubishi Gas Chemical(Tokyo, Japan).
                      </p>
                  </div>
              </div>
-             <div class="section our-team" id="our-team">
+             <div class="section our-team" id="bacteria_pcr_of_msr-1">
                  <div class="container1">
-                     <h2>Reference</h2>
+                     <h2>Bacterial PCR of MSR-1</h2>
                      <p>
-                         [1]. Ahmadzadeh, M., Farshdari, F., Nematollahi, L. et al. Anti-HER2 scFv Expression in Escherichia coli SHuffle<sup>®</sup>T7 Express Cells: Effects on Solubility and Biological Activity. Mol Biotechnol 62, 18–30 (2020).
+                         Because magnetotactic bacteria grew too slowly to form a visible colony, we tent to demonstrate that it was truly grown in the medium. The liquid medium was centrifuged at 12000 rpm for 10 minutes and the sediment was used for bacterial PCR using specific primers designed from the 16srDNA of <i>Magnetospirillum gryphiswaldense</i>, <i>E.coli</i> and <i>Agrobacterium</i> were used in the bacterial PCR as the negative control.
                      </p>
                      <br>
                      <br>
                  </div>
              </div>
+             <div class="section our-team" id="references">
+                <div class="container1">
-         </div>
+                    <h2>References</h2>
+                    <p>
+                        [1]. Ahmadzadeh, M., Farshdari, F., Nematollahi, L., Behdani, M., & Mohit, E. (2020). Anti-HER2
+                        scFv Expression in Escherichia coli SHuffle<sup>®</sup>T7 Express Cells: Effects on Solubility
+                        and Biological Activity. <i>Molecular biotechnology, 62</i>(1), 18–30.
+                        https://doi.org/10.1007/s12033-019-00221-2
+                    </p>
+                    <br>
+                    <br>
+                    <br>
+                    <br>
+                    <br>
+                </div>
+             </div>
+         </div>
+    </div>
 </div>
+<div class="footer" style="background-image: url('https://static.igem.org/mediawiki/2020/7/7f/T--ZJU-China--wiki_index_foot.png');background-size:100% 100%;">
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+    </div>
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-<script type="text/javascript" src="https://2020.igem.org/wiki/index.php?title=Template:ZJU-China/jscustom1&action=raw&ctype=text/javascript"></script>
+    </script>
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+    <script>
-    var typed = new Typed('#typed', {
+        var typed = new Typed('#typed', {
-        stringsElement: '#typed-strings',
+            stringsElement: '#typed-strings',
-        typeSpeed: 100,
+            typeSpeed: 100,
-        backSpeed: 100,
+            backSpeed: 100,
-        loop: true,
+            loop: true,
-        smartBackspace: true,
+            smartBackspace: true,
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+    <script>
-    function topMao(target){
+        function topMao(target) {
-    $('html, body').animate({scrollTop: $(target).offset().top - 170}, 500);//130为锚点到距顶部的距离，500为执行时间
+            $('html, body').animate({ scrollTop: $(target).offset().top - 170 }, 500);//130为锚点到距顶部的距离，500为执行时间
-    return true;
+            return true;
-}
+        }
-</script>
+    </script>
-<script>
+    <script>
-    var topBegin=$("#accordion").offset().top;  //获取导航栏（class='positionMiddleNav'）离视口的高度
+        var topBegin = $("#accordion").offset().top;  //获取导航栏（class='positionMiddleNav'）离视口的高度
-　　　　$(window).scroll(function(){  //scroll事件
+        $(window).scroll(function () {  //scroll事件
-　　　　　　var win_top=$(this).scrollTop(); //获取滚动条位置
+            var win_top = $(this).scrollTop(); //获取滚动条位置
-　　　　　　var _top=$("#accordion").offset().top; //获取当前导航栏距视口高度
+            var _top = $("#accordion").offset().top; //获取当前导航栏距视口高度
-　　　　　　 console.log(win_top , 'aa');
+            console.log(win_top, 'aa');
-              console.log(_top , 'cc');
+            console.log(_top, 'cc');
-　　　　　　if(win_top>=_top-150){
+            if (win_top >= _top - 150) {
-　　　　　　　$("#accordion").css({position: "fixed",top:"10em"});
+                $("#accordion").css({ position: "fixed", top: "10em" });
-　　　　　　}
+            }
-　　　　　　if(win_top<topBegin){ //因为导航栏距视口高度在定位发生后是变化的 ，
+            if (win_top < topBegin) { //因为导航栏距视口高度在定位发生后是变化的 ，
-　　　　　　//所以当导航栏回到原位置时保持先前状态需要将滚动条位置与最先前的导航栏位置进行对比
+                //所以当导航栏回到原位置时保持先前状态需要将滚动条位置与最先前的导航栏位置进行对比
-　　　　　　　$("#accordion").css({position: "absolute"});
+                $("#accordion").css({ position: "absolute" });
-　　　　　　}
+            }
-　　　　})
+        })
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+    </script>
+    <script>
-<script>
+        function displaywindowsize() {
+            var w = document.documentElement.clientWidth;
+            var h = document.documentElement.clientHeight;
+            if (w < 1100 && w > 800) {
-    function displaywindowsize(){
+                $("#accordion").css({ left: "2%" });
-        var w = document.documentElement.clientWidth;
+                $("#accordion").css({ display: "block" });
-        var h = document.documentElement.clientHeight;
+            }
-        if(w<1100&&w>800){
+            else if (w < 800) {
+                $("#accordion").css({ display: "none" });
-             $("#accordion").css({left:"2%"});
+             }
-            $("#accordion").css({display:"block"});
+            else {
+                $("#accordion").css({ left: "10%" });
+                $("#accordion").css({ display: "block" });
+            }
          }
-        else if(w<800){
-            $("#accordion").css({display:"none"});
-        }
-        else{
-            $("#accordion").css({left:"10%"});
-            $("#accordion").css({display:"block"});
-        }
-    }
-    window.addEventListener("resize",displaywindowsize);
+        window.addEventListener("resize", displaywindowsize);
-    displaywindowsize();
+        displaywindowsize();
-</script>
+    </script>

Difference between revisions of "Team:ZJU-China/Experiments"