iGEM 2023 — Team tokyotech


import { Layout } from "@/components/Layout";
import { MyHead } from "@/components/SEO";
import { ComingSoon } from "@/components/ComingSoon";
import { useCallback, useMemo, useState } from "react";
import { Cycle, Theme } from "@/components/pages/engneering";
import { CiteRefCtx } from "@/utils/useRefCtx";
import { Citable, CiteMap, ReferenceMap } from "@/types";
import { CiteRefList } from "@/components/DocComponents";
import styles from "@/styles/pages/engineering.module.scss";
import Image from "next/image";

const EngneeringCiteList: Array<Citable> = [];

// Theme1 Cycle 1
const T1C1 = () => {
  const D = () => {
    return (
      <div>
        <p>
          In iGEM 2022, TokyoTech attempted to establish infection detection
          cell lines by two methods: Electroporation and Lipofection. Infection
          detecting cells were co-transfected with linearized pCAGGS N-Cre DNA:
          <strong>
            <a href="https://parts.igem.org/Part:BBa_K4419025">BBa_K4419025</a>
          </strong>{" "}
          and linearized pMC1 neo-polyA (Fig.1). The selection with G418 was
          planned for the gene <em>Neo^R</em> from pMC1 neo-polyA, but
          transformants could not be obtained because the concentration of G418
          was too high. Therefore, we planned an experiment to determine the
          optimal G418 concentration to obtain transformants.
        </p>
        <figure>
          <Image
            src={
              "https://static.igem.wiki/teams/4828/wiki/pages/engineering/fig1.png"
            }
            alt={"fig:1"}
            height={100}
            width={100}
          />
          <figcaption>
            Fig.1 | How to create infection-detecting cells Linearized pCAGGS
            N-Cre and pMC1 neo-polyA are co-transfected by Electroporation. The
            gene circuit is then incorporated into the cell genome by random
            integration. The resulting transformants (infection-detecting cells)
            are selected by applying selection pressure with G418.
          </figcaption>
        </figure>
      </div>
    );
  };
  const B = () => {
    return (
      <div>
        <p>
          To determine the optimal G418 concentration to obtain transformants,
          PULSE was added to Vero cells in the Electroporator and cultured for 1
          week in medium graded for G418 concentration. (
          <a href="https://2023.igem.wiki/tokyotech/experiments">Experiments</a>{" "}
          221123~221207) The results showed that G418 900 µg/mL with 30%
          Confluency after 1 week was optimal for screening (Fig.2).
        </p>
        <figure>
          <Image
            src={
              "https://static.igem.wiki/teams/4828/wiki/pages/engineering/fig2.png"
            }
            alt={"fig2"}
            height={100}
            width={100}
          />
          <figcaption>
            Fig.2 | Vero cells cultured in DMEM+10%FBS+G418 as medium for 1 week
            G418 concentrations are 0, 100, 300, 600, 900, 1200, and 1500 µg/mL,
            respectively. The white scale bar represents 200 µm.
          </figcaption>
        </figure>
      </div>
    );
  };
  const T = () => {
    return (
      <div>
        <p>
          Electroporation of pCAGGS N-Cre and pMC1 neo-polyA (Both are
          straightened) with G418 900 µg/mL was performed again as determined in
          Build and selection with G418. (
          <a href="https://2023.igem.wiki/tokyotech/experiments">Experiments</a>{" "}
          221213-221225) Selections were made for 13 days, but cultured cells
          did not form colonies and no fluorescence was observed (Fig.3).
        </p>
        <figure>
          <Image
            src={
              "https://static.igem.wiki/teams/4828/wiki/pages/engineering/fig3.png"
            }
            alt={"fig3"}
            height={100}
            width={100}
          />
          <figcaption>
            Fig.3 | Photograph of Vero cells electroporated with pCAGGS N-Cre
            and pMC1 neo-polyA and normal Vero cells selected with G418 G418
            concentration was 900 µg/mL, selection was performed for 13 days.
            White scale bar is 200 µm.
          </figcaption>
        </figure>
      </div>
    );
  };
  const L = () => {
    return (
      <div>
        <p>
          We learned from this Test that the concentration of G418 is not the
          only thing inhibiting the selection of transformants. We also
          discovered that the resistance gene may not work or the DNA may not
          enter the cells.
        </p>
      </div>
    );
  };

  return (
    <Cycle
      cycleTitle={
        <>
          <h1 style={{ display: "inline" }}>Cycle 1: </h1>
          <span>Consideration of selection method</span>
        </>
      }
      design={<D />}
      build={<B />}
      test={<T />}
      learn={<L />}
    />
  );
};

const T1C2 = () => {
  const D = () => {
    return (
      <div>
        <p>
          In Cycle 1, we tried to obtain transformants by changing the
          concentration of G418 but failed. They also found that the resistance
          gene may not have worked, or the DNA may not have entered the cell.
          Therefore, we decided to reduce the difficulty of transfection by
          changing the method to transfection of one type of DNA instead of
          co-transfection of pCAGGS N-Cre and pMC1 neo-polyA (both are
          linearized).
        </p>
        <p>
          This means that Parts{" "}
          <a href="http://parts.igem.org/Part:BBa_K4419025">BBa_K4419025</a> has
          been improved: its promoter has been changed from a CAG promoter to a
          CMV promoter and a neomycin/kanamycin resistance gene has been added
          to the gene circuit.{" "}
          <a href="http://parts.igem.org/Part:BBa_K4828018">BBa_K4828018</a> was
          added to the Parts Registry.
        </p>
      </div>
    );
  };
  const B = () => {
    return (
      <div>
        <p>
          As stated in the Design, in order to use a single DNA transfection
          method, we considered that the Neo-resistant gene should be present in
          the plasmid backbone that incorporates the genetic circuit of the
          infection-detecting cell. Therefore, we used pCMV N-Cre, a product in
          the process of making pCAGGS N-Cre. pCMV N-Cre has pIRES2-EGFP as its
          backbone and has Neo^R, so only one type of DNA is needed for
          transformation (Fig.4) .
        </p>
        <figure>
          <img
            src="https://static.igem.wiki/teams/4828/wiki/pages/engineering/fig4.png"
            alt="Fig.4 How to create infection-detecting cells2
    Linearized pCMV N-Cre is transfected by Electroporation. The gene circuit is then incorporated into the cell genome by random integration. The resulting transformants (infection-detecting cells) are selected by applying selection pressure with G418."
          />
          <figcaption>
            Fig.4 How to create infection-detecting cells2 Linearized pCMV N-Cre
            is transfected by Electroporation. The gene circuit is then
            incorporated into the cell genome by random integration. The
            resulting transformants (infection-detecting cells) are selected by
            applying selection pressure with G418.
          </figcaption>
        </figure>
      </div>
    );
  };
  const T = () => {
    return (
      <div>
        <p>
          We electroporated Linearized pCMV N-Cre into Vero as described in
          Build and performed selection with G418 900 µg/mL as required in Cycle
          1. (
          <a href="https://2023.igem.wiki/tokyotech/experiments">Experiments</a>{" "}
          230221-230306) However, no transformants were obtained (Fig.5).
        </p>
        <figure>
          <img
            src="https://static.igem.wiki/teams/4828/wiki/pages/engineering/fig5.png"
            alt="Fig.5 Photograph of Vero cells electroporated with pCMV N-Cre and normal Vero cells selected with G418
    G418 concentration was 900 µg/mL, selection was performed for 13 days. White scale bar is 200 µm."
          />
          <figcaption>
            Fig.5 Photograph of Vero cells electroporated with pCMV N-Cre and
            normal Vero cells selected with G418 G418 concentration was 900
            µg/mL, selection was performed for 13 days. White scale bar is 200
            µm.
          </figcaption>
        </figure>
      </div>
    );
  };
  const L = () => {
    return (
      <div>
        We realized that co-transfection was not the root of the problem with
        this Test. Since the fluorescence of this Test showed little green
        fluorescence, we could hypothesize that very little DNA was transformed
        into the Vero cells.
      </div>
    );
  };
  return (
    <Cycle
      cycleTitle={
        <>
          <h1 style={{ display: "inline", lineHeight: "2rem" }}>Cycle 2: </h1>
          <span style={{ lineHeight: "2rem" }}>Change of Plasmid backbone</span>
        </>
      }
      design={<D />}
      build={<B />}
      test={<T />}
      learn={<L />}
    />
  );
};
const T1C3 = () => {
  const D = () => {
    return (
      <div>
        <p>
          In Cycle 2, the plasmid backbone was changed to reduce the difficulty
          of transformation, but no transformants were obtained. However, we
          thought that Vero cells might contain little DNA, so we decided to
          review the conditions of Electroporation.
        </p>
      </div>
    );
  };
  const B = () => {
    return (
      <div>
        <p>
          As described in{" "}
          <a href="https://2023.igem.wiki/tokyotech/experiments">Experiments</a>{" "}
          230412, 230427, Linearized pCMV N-Cre under various PULSE conditions
          were electroporated and selected with G418 900 µg/mL. (
          <a href="https://2023.igem.wiki/tokyotech/experiments">Experiments</a>
          230412-230417, 230427-230430)
        </p>
        <p>
          We also stained some of the cells to which PULSE was added with trypan
          blue to see how many cells were killed by PULSE.
        </p>
      </div>
    );
  };
  const T = () => {
    return (
      <div>
        <p>
          The selection did not result in transformants. In addition, a few
          cells were stained with trypan blue immediately after PULSE was added.
        </p>
      </div>
    );
  };
  const L = () => {
    return (
      <div>
        <p>
          We were able to confirm by this test that PULSE did not work at all
          against Vero cells. We thought that this was due to the conditions of
          the Electroporator or Electroporation.
        </p>
      </div>
    );
  };
  return (
    <Cycle
      cycleTitle={
        <>
          <h1 style={{ display: "inline", lineHeight: "2rem" }}>Cycle 3: </h1>
          <span style={{ lineHeight: "2rem" }}>
            {" "}
            Considerations of Electroporation
          </span>
        </>
      }
      design={<D />}
      build={<B />}
      test={<T />}
      learn={<L />}
    />
  );
};
const T1C4 = () => {
  const D = () => {
    return (
      <div>
        <p>
          In Cycle 3 we discovered that there was some problem with our
          Electroporation. Therefore, we planned to change our transformation
          method. First of all, we considered changing the conditions of
          electroporation. However, we soon realized that it was not realistic
          to search for the optimal PULSE because there were too many conditions
          to consider. Next, we considered trying Lipofection. However, we gave
          up because of the high cost of the reagents. So we decided to consult
          Dr. Suzuki, who has been helping us with SRIP production experiments.
        </p>
      </div>
    );
  };
  const B = () => {
    return (
      <div>
        <p>
          After consulting with Dr. Suzuki, he recommended using PEI:
          polyethylenimine, which is inexpensive and often used for
          transfection, and gave us some PEI (Fig.6). (See{" "}
          <a href="https://2023.igem.wiki/tokyotech/human-practices">
            Human Practices
          </a>{" "}
          for details.) We immediately performed transfection using PEI. (
          <a href="https://2023.igem.wiki/tokyotech/experiments">Experiments</a>{" "}
          230901, 230905)
        </p>
        <figure>
          <img
            src="https://static.igem.wiki/teams/4828/wiki/pages/engineering/fig6.png"
            alt="Fig.6 iGEM TokyoTech members with Dr. Suzuki and Dr. Matsuda from National Institute of Infectious Diseases"
          />
          <figcaption>
            Fig.6 iGEM TokyoTech members with Dr. Suzuki and Dr. Matsuda from
            National Institute of Infectious Diseases
          </figcaption>
        </figure>
      </div>
    );
  };
  const T = () => {
    return (
      <div>
        <p>
          The high fluorescence intensity, which was not seen in
          Electroporation, was confirmed. However, the PEI-based method
          transformed cyclic pCMV N-Cre instead of linearized pCMV N-Cre
          (Fig.7).
        </p>
        <figure>
          <img
            src="https://static.igem.wiki/teams/4828/wiki/pages/engineering/fig7.png"
            alt="Fig.7
    A: pCMV N-Cre plasmid map
    B: SRIP infection detecting cells
    Vero transfected with pCMV N-Cre by using  PEI: Polyethylenimine"
          />
          <figcaption>
            Fig.7 A: pCMV N-Cre plasmid map B: SRIP infection detecting cells
            Vero transfected with pCMV N-Cre by using PEI: Polyethylenimine
          </figcaption>
        </figure>
      </div>
    );
  };
  const L = () => {
    return (
      <div>
        <p>
          PEI can successfully transform the cells, indirectly proving that
          there is a problem with the electroporation conditions as per Learn in
          Cycle 3. Although PEI involves introducing a circular plasmid, it is
          still necessary to aim for random integration into the cell genome by
          transforming linear DNA to isolate transformants, i.e., to establish
          cell lines.
        </p>
      </div>
    );
  };
  return (
    <Cycle
      cycleTitle={
        <>
          <h1 style={{ display: "inline", lineHeight: "2rem" }}>Cycle 4: </h1>
          <span style={{ lineHeight: "2rem" }}>
            Change of transformation method
          </span>
        </>
      }
      design={<D />}
      build={<B />}
      test={<T />}
      learn={<L />}
    />
  );
};
const T2C1 = () => {
  const D = () => {
    return (
      <div>
        First, we planned an experiment to create a Growth Curve by seeding
        C6/36 cells in 96-well plates and counting the number of C6/36 cells
        using WST-8. However, the cells did not grow well due to bacterial
        contamination. Therefore, we decided to examine the cause of
        contamination first.
      </div>
    );
  };
  const B = () => {
    return (
      <div>
        The medium used was placed in an empty dish and incubated at 37°C
        overnight to ensure that the medium used was not contaminated.
      </div>
    );
  };
  const T = () => {
    return <div>It was confirmed that the medium was contaminated.</div>;
  };
  const L = () => {
    return <div>Penicillin was added to the next medium used.</div>;
  };
  return (
    <Cycle
      cycleTitle={
        <>
          <h1 style={{ display: "inline", lineHeight: "2rem" }}>Cycle 1: </h1>
          <span style={{ lineHeight: "2rem" }}>
            Elucidation of the cause of contamination1
          </span>
        </>
      }
      design={<D />}
      build={<B />}
      test={<T />}
      learn={<L />}
    />
  );
};
const T2C2 = () => {
  const D = () => {
    return (
      <div>
        We tried to start the experiment again with penicillin-containing medium
        as improved in Cycle 1, but again contamination was observed. We decided
        to further investigate the cause of the contamination.
      </div>
    );
  };
  const B = () => {
    return (
      <div>
        We thought that the experimenter&apos;s poor technique was causing the
        contamination, so we changed the method of handling the cells from a 1
        mL stripper to a dropper.
      </div>
    );
  };
  const T = () => {
    return (
      <div>
        No contamination was observed when cells were seeded with the dropper.
      </div>
    );
  };
  const L = () => {
    return (
      <div>
        We learned that the manipulation at hand plays a major role in
        contamination and that the skill of the experimenter is essential for
        the success of the experiment.
      </div>
    );
  };
  return (
    <Cycle
      cycleTitle={
        <>
          <h1 style={{ display: "inline", lineHeight: "2rem" }}>Cycle 2: </h1>
          <span style={{ lineHeight: "2rem" }}>
            Elucidation of the cause of contamination2
          </span>
        </>
      }
      design={<D />}
      build={<B />}
      test={<T />}
      learn={<L />}
    />
  );
};
const T3C1 = () => {
  const D = () => {
    return (
      <div>
        <p>
          As described in{" "}
          <a href="https://2023.igem.wiki/tokyotech/experiments">Experiments</a>
          230206, we cut out the nluc gene using restriction enzymes and ligated
          the C-Cre gene as an insert. However, when we transformed the ligation
          product, no colonies grew. Since we were concerned about the low
          amount of vector during the experiment, we decided to extract more
          plasmids from E. coli because we thought it was necessary to prepare a
          larger amount of plasmids.
        </p>
      </div>
    );
  };
  const B = () => {
    return (
      <div>
        <p>
          Since pCMV YF-C-Cre-rep is a low-copy plasmid, we added
          chloramphenicol to E. coli culture medium in the logarithmic growth
          phase. This resulted in a 3-fold increase in the amount of plasmid
          compared to normal (Fig.8).(
          <a href="https://2023.igem.wiki/tokyotech/experiments">Experiments</a>
          230519)
        </p>
        <figure>
          <img
            src="https://static.igem.wiki/teams/4828/wiki/pages/engineering/fig8.png"
            alt="Fig.8 Analysis by agarose gel electro phoresis about pCMV YF-nluc-rep plasmid prepartaion
    A: 0.6% agarose gel image, loaded with pCMV YF-nluc-rep. Left: Plasmid extracted from E. coli cultured in LB medium without chloramphenicol. Right: plasmid extracted from E. coli cultured in LB medium with 25 µg/mL chloramphenicol. ×4, ×2, ×1, ×1/2, and ×1/4 indicate how much plasmid solution was added based on ×1.
    B: pCMV YF-nluc-rep plasmid map"
          />
          <figcaption>
            Fig.8 Analysis by agarose gel electro phoresis about pCMV
            YF-nluc-rep plasmid prepartaion A: 0.6% agarose gel image, loaded
            with pCMV YF-nluc-rep. Left: Plasmid extracted from E. coli cultured
            in LB medium without chloramphenicol. Right: plasmid extracted from
            E. coli cultured in LB medium with 25 µg/mL chloramphenicol. ×4, ×2,
            ×1, ×1/2, and ×1/4 indicate how much plasmid solution was added
            based on ×1. B: pCMV YF-nluc-rep plasmid map
          </figcaption>
        </figure>
      </div>
    );
  };
  const T = () => {
    return (
      <div>
        <p>
          A sufficient amount of plasmid was digested with SnaB I, the next
          step. However, there was still some residue, so another overnight
          restriction enzyme treatment was performed. However, there were still
          remnants (Fig. 9).(
          <a href="https://2023.igem.wiki/tokyotech/experiments">Experiments</a>
          230607)
        </p>
        <figure>
          <img
            src="https://static.igem.wiki/teams/4828/wiki/pages/engineering/fig9.png"
            alt="Fig.9 Analysis by agarose gel electro phoresis about pCMV YF-nluc-rep SnaB I digestion
    A: 0.6% agarose gel image, loaded with extracted pCMV YF-luc-rep from E.coli and pCMV YF-luc-rep digested with SnaB I.
    B: pCMV YF-luc-rep plasmid map."
          />
          <figcaption>
            Fig.9 Analysis by agarose gel electro phoresis about pCMV
            YF-nluc-rep SnaB I digestion A: 0.6% agarose gel image, loaded with
            extracted pCMV YF-luc-rep from E.coli and pCMV YF-luc-rep digested
            with SnaB I. B: pCMV YF-luc-rep plasmid map.
          </figcaption>
        </figure>
      </div>
    );
  };
  const L = () => {
    return (
      <div>
        <p>
          From the electrophoresis results of Test, we assumed that there was
          some reason why the plasmid could not be digested by restriction
          enzymes.
        </p>
      </div>
    );
  };
  return (
    <Cycle
      cycleTitle={
        <>
          <h1 style={{ display: "inline", lineHeight: "2rem" }}>Cycle 1: </h1>
          <span style={{ lineHeight: "2rem" }}>
            Plasmid Construction with Restriction Enzymes and Ligases
          </span>
        </>
      }
      design={<D />}
      build={<B />}
      test={<T />}
      learn={<L />}
    />
  );
};
const T3C2 = () => {
  const D = () => {
    return (
      <div>
        <p>
          We found that the plasmid used in Cycle 1 was not digestible by the
          restriction enzyme. In response to this, we consulted with our PI and
          considered the possibility that the prolonged incubation of E. coli
          may have altered the properties of the plasmid.
        </p>
      </div>
    );
  };
  const B = () => {
    return (
      <div>
        <p>
          To achieve what we discussed in Design, we performed plasmid
          extraction by reducing the incubation time of mass culture of E. coli
          transformed with pCMV YF-nluc-rep to less than 12 hours.
        </p>
      </div>
    );
  };
  const T = () => {
    return (
      <div>
        <p>
          The plasmid extracted with Build was treated with restriction enzyme
          with SnaB I, but the remainder of the cut was still visible (Fig.
          10).(
          <a href="https://2023.igem.wiki/tokyotech/experiments">Experiments</a>
          230719)
        </p>
        <figure>
          <img
            src="https://static.igem.wiki/teams/4828/wiki/pages/engineering/fig10.png"
            alt="Fig.10 Analysis by agarose gel electro phoresis about pCMV YF-nluc-rep (Short-Time Culture) SnaB I digestion
    A: 0.6% agarose gel image, loaded with extracted pCMV YF-nluc-rep from E.coli and pCMV YF-luc-rep digested with SnaB I
    B: pCMV YF-luc-rep plasmid map"
          />
          <figcaption>
            Fig.10 Analysis by agarose gel electro phoresis about pCMV
            YF-nluc-rep (Short-Time Culture) SnaB I digestion A: 0.6% agarose
            gel image, loaded with extracted pCMV YF-nluc-rep from E.coli and
            pCMV YF-luc-rep digested with SnaB I B: pCMV YF-luc-rep plasmid map
          </figcaption>
        </figure>
      </div>
    );
  };
  const L = () => {
    return (
      <div>
        <p>
          It was found that prolonged incubation was not the direct cause of the
          inability to process with the limiting enzyme.
        </p>
      </div>
    );
  };
  return (
    <Cycle
      cycleTitle={
        <>
          <h1 style={{ display: "inline", lineHeight: "2rem" }}>Cycle 2-1: </h1>
          <span style={{ lineHeight: "2rem" }}>
            Consideration of incubation time for restriction enzyme treatment
          </span>
        </>
      }
      design={<D />}
      build={<B />}
      test={<T />}
      learn={<L />}
    />
  );
};
const T3C3 = () => {
  const D = () => {
    return (
      <div>
        <p>
          In Cycle 1, we found that the plasmid used could not be digested by
          the restriction enzyme. In response to this, we consulted with our
          advisor and thought that the restriction enzyme might not be working
          well because the plasmid was taking on a higher-order structure.
          Therefore, we decided to perform heat shock to resolve the
          higher‐order structure of the plasmid.
        </p>
      </div>
    );
  };
  const B = () => {
    return (
      <div>
        <p>
          pCMV YF-nluc-rep (R: SnaB I) was incubated at 65°C for 5 min and
          cooled rapidly in an attempt to resolve the higher-order structure.
          Restriction enzyme treatment was later performed again with SnaB I.
        </p>
      </div>
    );
  };
  const T = () => {
    return (
      <div>
        <figure>
          <img
            src="https://static.igem.wiki/teams/4828/wiki/pages/engineering/fig11.png"
            alt="Fig.11 Analysis by agarose gel electrophoresis about pCMV YF-nluc-rep (R: SnaB I, Heat-shocked) SnaB I digestion
    A: 0.6% agarose gel image, loaded with extracted pCMV YF-nluc-rep (R: SnaB I) and pCMV YF-luc-rep (R: SnaB I, Heat-shocked) digested with SnaB I
    B: pCMV YF-luc-rep plasmid map"
          />
          <figcaption>
            Fig.11 Analysis by agarose gel electrophoresis about pCMV
            YF-nluc-rep (R: SnaB I, Heat-shocked) SnaB I digestion A: 0.6%
            agarose gel image, loaded with extracted pCMV YF-nluc-rep (R: SnaB
            I) and pCMV YF-luc-rep (R: SnaB I, Heat-shocked) digested with SnaB
            I B: pCMV YF-luc-rep plasmid map
          </figcaption>
        </figure>
      </div>
    );
  };
  const L = () => {
    return (
      <div>
        <p>
          Higher-order structures are not the direct cause of the breakage
          remnant.
        </p>
      </div>
    );
  };
  return (
    <Cycle
      cycleTitle={
        <>
          <h1 style={{ display: "inline", lineHeight: "2rem" }}>Cycle 2-2: </h1>
          <span style={{ lineHeight: "2rem" }}>Plasmid heat shock</span>
        </>
      }
      design={<D />}
      build={<B />}
      test={<T />}
      learn={<L />}
    />
  );
};
const T3C4 = () => {
  const D = () => {
    return (
      <div>
        <p>
          Cycle 2-1 and 2-2 were not able to eliminate the remaining pieces.
          Therefore, we planned to purify only the restriction enzyme-digested
          portions and use them for downstream.
        </p>
      </div>
    );
  };
  const B = () => {
    return (
      <div>
        <p>
          The completely broken DNA was purified and used in a later ligation
          reaction for transformation.(
          <a href="https://2023.igem.wiki/tokyotech/experiments">Experiments</a>
          230822-230824)
        </p>
      </div>
    );
  };
  const T = () => {
    return (
      <div>
        <p>
          Colonies grew, so small cultures and small preparations of plasmid
          were made for all colonies. The extracted plasmids were checked for
          per colony by restriction enzyme Xma I digestion, but there were none
          (Fig.12).(
          <a href="https://2023.igem.wiki/tokyotech/experiments">Experiments</a>
          230825-230901)
        </p>
        <figure>
          <img
            src="https://static.igem.wiki/teams/4828/wiki/pages/engineering/fig12.png"
            alt="Fig.12 Analysis by agarose gel electro phoresis about pCMV YF-C-Cre-rep colonies Xma I digestion
    A: 0.6% agarose gel image, loaded with extracted pCMV YF-C-Cre-rep from E.coli and them digested with Xma I (expressed as R)
    B: pCMV YF-C-Cre-rep plasmid map"
          />
          <figcaption>
            Fig.12 Analysis by agarose gel electro phoresis about pCMV
            YF-C-Cre-rep colonies Xma I digestion A: 0.6% agarose gel image,
            loaded with extracted pCMV YF-C-Cre-rep from E.coli and them
            digested with Xma I (expressed as R) B: pCMV YF-C-Cre-rep plasmid
            map
          </figcaption>
        </figure>
      </div>
    );
  };
  const L = () => {
    return (
      <div>
        <p>
          We had considered plasmid construction using restriction enzymes and
          ligase for Cycle 1-4, but considering the time remaining, we decided
          that we would be closer to completion if we tried a different
          approach.
        </p>
      </div>
    );
  };
  return (
    <Cycle
      cycleTitle={
        <>
          <h1 style={{ display: "inline", lineHeight: "2rem" }}>Cycle 3: </h1>
          <span style={{ lineHeight: "2rem" }}>
            Use of products separated by electrophoresis
          </span>
        </>
      }
      design={<D />}
      build={<B />}
      test={<T />}
      learn={<L />}
    />
  );
};
const T3C5 = () => {
  const D = () => {
    return (
      <div>
        <p>
          Consider plasmid constructions without restriction enzymes and
          ligases.
        </p>
      </div>
    );
  };
  const B = () => {
    return (
      <div>
        <p>
          Inverse PCR was used to amplify all but the nluc gene of pCMV
          YF-nluc-rep, and the C-Cre gene was incorporated by TaKaRa&#39;s
          InFusion to produce pCMV YF-C-Cre-rep, which was transformed into
          E.coli.(
          <a href="https://2023.igem.wiki/tokyotech/experiments">Experiments</a>
          230906-230921)
        </p>
      </div>
    );
  };
  const T = () => {
    return (
      <div>
        <p>
          Colonies were grown and colony PCR was performed. The colonies per
          colony were supposed to show a band at 383 b. However, all the bands
          that were seen appeared at 563 b or higher. However, the bands that
          were seen were all at 563 b or higher. Considering that the primers
          used in this colony PCR annealed to pCMV YF-nluc-rep and produced a
          DNA fragment of 599 b, it is likely that all the colonies that emerged
          this time had the original pCMV YF-nluc-rep vector (Fig. 13).(
          <a href="https://2023.igem.wiki/tokyotech/experiments">Experiments</a>
          230922){" "}
        </p>
        <figure>
          <img
            src="https://static.igem.wiki/teams/4828/wiki/pages/engineering/fig13.png"
            alt="Fig.13 Analysis by agarose gel electro phoresis about pCMV YF-C-Cre-rep colony PCR
    A: 0.6% agarose gel image, loaded with colony PCR product
    B: pCMV YF-C-Cre-rep plasmid map"
          />
          <figcaption>
            Fig.13 Analysis by agarose gel electro phoresis about pCMV
            YF-C-Cre-rep colony PCR A: 0.6% agarose gel image, loaded with
            colony PCR product B: pCMV YF-C-Cre-rep plasmid map
          </figcaption>
        </figure>
      </div>
    );
  };
  const L = () => {
    return (
      <div>
        <p>
          We were unable to complete the plasmid construction in InFusion
          because the PCR products were not purified by the method of cutting
          them out of the gel, which we believe resulted in contamination of the
          large source plasmid. Therefore, we believe that the method of gel
          extraction of the PCR product would allow us to successfully complete
          the plasmid construction in InFusion.
        </p>
      </div>
    );
  };
  return (
    <Cycle
      cycleTitle={
        <>
          <h1 style={{ display: "inline", lineHeight: "2rem" }}>Cycle 4: </h1>
          <span style={{ lineHeight: "2rem" }}>
            Investigation of Inverse PCR and InFusion
          </span>
        </>
      }
      design={<D />}
      build={<B />}
      test={<T />}
      learn={<L />}
    />
  );
};
const WetCycles = () => {
  return (
    <>
      <Theme
        themeName={
          <h1>
            Investigation of a method for generating infection-detecting cells
          </h1>
        }
        description={
          <p>
            CFNT: Cell Fluorescence Neutralization Test measures the amount of
            neutralizing antibodies in the subject&#39;s serum as described in{" "}
            <a href="https://2023.igem.wiki/tokyotech/description">
              Description
            </a>{" "}
            and <a href="https://2023.igem.wiki/tokyotech/design">Design</a>. As
            described in{" "}
            <a href="https://2023.igem.wiki/tokyotech/description">
              Description
            </a>{" "}
            and <a href="https://2023.igem.wiki/tokyotech/design">Design</a>,
            CFNT measures the number of neutralizing antibodies in the
            subject&#39;s serum. Since the number of neutralizing antibodies is
            quantified by the ratio of green to red fluorescence,
            infection-detecting cells are one of the most important elements in
            CFNT. The quality of infection detection cells should be maintained
            to guarantee the sensitivity and specificity of CFNT when it is
            implemented in society in the future. In other words,
            infection-detecting cell lines should be established, and we have
            performed several DBTL cycles for this purpose.
          </p>
        }
        cycleArray={[
          <T1C1 key={1} />,
          <T1C2 key={2} />,
          <T1C3 key={3} />,
          <T1C4 key={4} />,
        ]}
      />
      <Theme
        themeName={
          <h1>
            Investigation of a method for culturing C6/36 cells without an
            incubator
          </h1>
        }
        description={
          <p>
            As stated in the{" "}
            <a href="https://2023.igem.wiki/tokyotech/description">
              Description
            </a>{" "}
            and <a href="https://2023.igem.wiki/tokyotech/design">Design</a>,
            the CFNT: Cell Fluorescence Neutralization Test measures the amount
            of neutralizing antibodies in the subject&#39;s serum. The amount of
            neutralizing antibodies is quantified by the ratio of green to red
            fluorescence, making the infection-detecting cells one of the most
            important components of the CFNT. In addition, as shown in the{" "}
            <a href="https://2023.igem.wiki/tokyotech/design">Design</a> and{" "}
            <a href="https://2023.igem.wiki/tokyotech/proof-of-concept">
              Proof of Concept
            </a>
            , the cost of introducing CFNTs will be reduced if
            infection-detecting cells can be generated from mosquito C6/36 cells
            and cultured without an incubator. Therefore, we experimented by
            running a DBTL cycle to culture mosquito cells, C6/36 cells, without
            the maintenance of a 5% carbon dioxide concentration.
          </p>
        }
        cycleArray={[<T2C1 key={1} />, <T2C2 key={2} />]}
      />
      <Theme
        themeName={<h1>Preparation of pCMV YF-C-Cre-rep</h1>}
        description={
          <div>
            <p>
              CFNT: Cell Fluorescence Neutralization Test measures the amount of
              neutralizing antibodies in a subject&#39;s serum as described in{" "}
              <a href="https://2023.igem.wiki/tokyotech/description">
                Description
              </a>{" "}
              and <a href="https://2023.igem.wiki/tokyotech/design">Design</a>.
              As described in{" "}
              <a href="https://www.notion.so/4a9eb7e02ad14680992047f20d7a8553?pvs=21">
                Description
              </a>{" "}
              and <a href="https://2023.igem.wiki/tokyotech/design">Design</a>,
              it measures the amount of neutralizing antibodies in the
              subject&#39;s serum. The amount of neutralizing antibodies is
              quantified by the ratio of green to red fluorescence.
              Infection-detecting cells that normally show green fluorescence
              become red fluorescent when infected with SRIP: Single-round
              Infectious Particle, which is similar to DENV: Dengue virus. In
              other words, whether or not SRIP is neutralized by antibodies in
              the serum is determined by the color of the fluorescence, and the
              ratio of green to red fluorescence intensity is used to determine
              the anti-DENV neutralizing antibody titer. Therefore, SRIP is one
              of the most important components of CFNT.
            </p>
            <p>
              For the SRIP preparation, we replaced the <em>nluc</em> gene of
              pCMV YF-nluc-rep provided by Dr. Suzuki with the <em>C-Cre</em>{" "}
              gene to construct pCMV YF-C-Cre-rep (for details, see{" "}
              <a href="https://2023.igem.wiki/tokyotech/design">Design</a>),
              starting with the construction of pCMV YF-C-Cre-rep (see{" "}
              <a href="https://2023.igem.wiki/tokyotech/design">design</a> for
              details). We then ran multiple DBTL cycles to complete this
              construction.
            </p>
          </div>
        }
        cycleArray={[
          <T3C1 key={1} />,
          <T3C2 key={2} />,
          <T3C3 key={3} />,
          <T3C4 key={4} />,
          <T3C5 key={5} />,
        ]}
      />
    </>
  );
};

const DryCycles = () => {
  return (
    <>
      <Theme
        themeName={<h1>Dry</h1>}
        description={<p>This is some description</p>}
        cycleArray={[<T1C1 key={1} />, <T1C2 key={2} />]}
      />
    </>
  );
};

const Engineering = () => {
  const [opened, setOpened] = useState<"Wet" | "Dry">("Wet");
  const [map, setMap] = useState<{ cite: CiteMap; ref: ReferenceMap }>({
    cite: {},
    ref: {},
  });
  const memoSetMap = useCallback(
    (
      args:
        | { cite: CiteMap; ref: ReferenceMap }
        | ((prev: { cite: CiteMap; ref: ReferenceMap }) => {
            cite: CiteMap;
            ref: ReferenceMap;
          })
    ) => {
      setMap(args);
    },
    []
  );
  const value = useMemo(
    () => ({
      map: map,
      setMap: memoSetMap,
    }),
    [map, memoSetMap]
  );
  return (
    <>
      <MyHead title="Engineering Success" />
      <Layout>
        <p
          style={{
            width: "70%",
            margin: "60px auto",
            lineHeight: "1.5",
            fontSize: "24px",
            padding: "0 9px",
          }}
        >
          In order to complete Project &quot;STAND UP&quot;, we have been
          working on the development in a more engineering way by implementing
          the Design, Build, Test, Learn Cycle (: DBTL Cycle). In this page, we
          will introduce how we have tackled various problems by implementing
          the DBTL Cycle from the aspects of Wet and Dry.
        </p>

        <CiteRefCtx.Provider value={value}>
          <div className={`${styles.theme__container}`}>
            <div className={`${styles.button__wrapper}`}>
              <button
                onClick={() => {
                  setOpened("Wet");
                }}
              >
                Wet
              </button>
              <button
                onClick={() => {
                  setOpened("Dry");
                }}
              >
                Dry
              </button>
            </div>
            <div>{opened === "Wet" ? <WetCycles /> : <DryCycles />}</div>
            <div style={{ padding: "2rem 10%" }}>
              <CiteRefList citeList={EngneeringCiteList} />
            </div>
          </div>
        </CiteRefCtx.Provider>
      </Layout>
    </>
  );
};

export default Engineering;

import { Layout } from "@/components/Layout";
import { MyHead } from "@/components/SEO";
import { DocLayout } from "@/components/DocLayout";
import {
  DocImg,
  H1,
  H2,
  Ref,
  P,
  PaperSection,
  H3,
} from "@/components/DocComponents";
import { HupraToggleTab } from "@/components/pages/education";
const Experiments = () => {
  return (
    <>
      <MyHead title="Experiments" />
      <Layout>
        <DocLayout>
          <PaperSection>
            <P>
              This page contains important Experiment methods in PDF format. In
              particular, the experimental methods used for the Model results
              are presented in text form.
            </P>
          </PaperSection>
          <PaperSection>
            <H1 refId="h1:methods-of-experiments">Methods of Experiments</H1>
            <div
              style={{
                display: "flex",
                alignItems: "center",
                justifyContent: "center",
              }}
            >
              <embed
                src="https://static.igem.wiki/teams/4828/wiki/pages/experiments/protocols.pdf"
                style={{ width: "100%", height: "50rem", maxHeight: "90vh" }}
              />
            </div>
          </PaperSection>
          <PaperSection>
            <H1 refId="h1:Methods-for-Dry-lab-Results">
              Methods for Dry-lab Results
            </H1>
            <H2 refId="h2:Measurement-of-Infectivity">
              Measurement of Infectivity Titers (IU) Using Dr. Suzuki&apos;s
              SRIP Method
            </H2>
            <H3 refId="h3:Production-of-SRIP-Using-HEK293T-Cells">
              Production of SRIP Using HEK293T Cells
            </H3>
            Overview: Transfection of Appropriate Plasmids into HEK293T Cells
            and Collection of SRIP
            <HupraToggleTab tabTitle={<>Methods</>}>
              <ul>
                <li>HEK293T Transfection</li>
              </ul>
              <p>Materials：</p>
              <p>pCMV YF-nluc-rep(1μg/μL)、pCAG D1-YG1-prME(500ng/μL)、</p>
              <p>　　　 pCAG YF-C(1μg/μL)</p>
              <p>Method：</p>
              <ol>
                <li>
                  Add 1 mL of Opti-MEM, 25μL of PEI, 2.5μL of pCMV YF-nluc-rep,
                  2.5μL of pCAG D1-YG1-prME, and 1.25μL of pCAG YF-C to a single
                  Eppendorf tube, making the total volume slightly over 1 mL.
                  Allow the mixture to sit for 15 minutes.
                </li>
                <li>
                  After the incubation period, add the mixture to HEK293T cells
                  in a P10+8 plate and incubate for 5 to 6 hours.
                </li>
                <li>After this time, replace the culture medium.</li>
                <li>Incubate the culture overnight.</li>
                <li>Collection of SRIP from HEK293T Cells (Day 1-9)</li>
              </ol>
              <p>Method：</p>
              <p>Day1</p>
              <ol>
                <li>
                  Harvest the supernatant from the culturing HEK293T cells.
                </li>
                <li>
                  Sterilize the collected supernatant using a 0.45μm filter.
                </li>
                <li>Store the filtered supernatant at -80°C.</li>
                <li>
                  Add 10 mL of warmed culture medium to the dish where the cells
                  have adhered and incubate at 37°C.
                </li>
              </ol>
              <p>Days 2-9</p>
              <ol>
                <li>
                  Process the culture medium from the HEK293T cells in the same
                  manner as on Day 1.
                </li>
                <li>
                  Store the collected and filtered culture medium at -80°C.
                </li>
                <li>
                  Add 10 mL of warmed culture medium to the dish where the cells
                  have adhered.
                </li>
                <li>Add 1M HEPES and incubate at 37°C.</li>
              </ol>
            </HupraToggleTab>
            <H3 refId="h3:Measurement-of-SRIP-Infectivity-Titer-(IU)">
              Measurement of SRIP Infectivity Titer (IU)
            </H3>
            Overview: Infect Vero cells with SRIP and Measure the Infectivity
            Titer (IU) of SRIP
            <HupraToggleTab tabTitle={<>Methods</>}>
              <ul>
                <li>
                  Part1:
                  <ol>
                    <li>Add 6 mL of culture medium to a centrifuge tube.</li>
                    <li>Sterilize the medium using a 0.45μm filter.</li>
                    <li>
                      Aliquot 500μL of the sterilized medium into individual
                      Eppendorf tubes.
                    </li>
                    <li>
                      Prepare 10-fold and 100-fold dilutions of the aliquoted
                      medium.
                    </li>
                    <li>
                      Remove the culture medium from the 96-well plate where
                      Vero cells have been seeded.
                    </li>
                    <li>
                      Add 100μL of each of the diluted culture media to the
                      respective wells.
                    </li>
                    <li>
                      After 5 to 6 hours, replace the culture medium in the
                      96-well plate.
                    </li>
                    <li>Incubate the plate at 37°C for 3 days.</li>
                  </ol>
                </li>
                <li>
                  part2:
                  <ol>
                    <li>
                      Wash the Vero P20 cells in the 96-well plate that have
                      been infected with SRIP using 50μL per well of PBS
                      (Phosphate-Buffered Saline).
                    </li>
                    <li>Allow the plate to air-dry for 1 hour.</li>
                    <li>
                      Add 100μL per well of cold acetone-methanol solution
                      (1:1=v/v) and incubate at 4°C for 20 minutes.
                    </li>
                    <li>Wash the cells again using 50μL per well of PBS.</li>
                    <li>
                      Add 50μL per well of Blocking One and let it sit at room
                      temperature for 30 minutes.
                    </li>
                    <li>
                      Wash the cells once more using 50μL per well of PBS.
                    </li>
                    <li>
                      Add 50μL per well of Anti YF-NS1 Protein, Rabbit Poly at a
                      1/2000 dilution and incubate overnight at 4°C.
                    </li>
                  </ol>
                </li>
                <li>
                  part3:
                  <ol>
                    <li>
                      Wash the Vero P20 cells in the 96-well plate that have
                      been infected with SRIP using 50μL/well of PBS
                      (Phosphate-Buffered Saline).
                    </li>
                    <li>Allow the plate to air-dry for 1 hour.</li>
                    <li>
                      Add 100μL/well of cold acetone-methanol solution (1:1=v/v)
                      and incubate at 4°C for 20 minutes.
                    </li>
                    <li>Wash the cells again using 50μL/well of PBS.</li>
                    <li>
                      Add 50μL/well of Blocking One and let it sit at room
                      temperature for 30 minutes.
                    </li>
                    <li>Wash the cells once more using 50μL/well of PBS.</li>
                    <li>
                      Add 50μL/well of Anti YF-NS1 Protein, Rabbit Poly at a
                      1/2000 dilution and incubate overnight at 4°C.
                    </li>
                  </ol>
                </li>
              </ul>
            </HupraToggleTab>
          </PaperSection>
        </DocLayout>
      </Layout>
    </>
  );
};

export default Experiments;

import { Layout } from "@/components/Layout";
import { MyHead } from "@/components/SEO";
import { DocLayout } from "@/components/DocLayout";
import {
  DocImg,
  H1,
  H2,
  Ref,
  P,
  PaperSection,
  H3,
  CiteRef,
  CiteRefList,
} from "@/components/DocComponents";
import Link from "next/link";
import { Citable } from "@/types";

const citeList: Array<Citable> = [
  {
    type: "row",
    citeId: "1",
    citeNumber: "1",
    content: (
      <>
        Yamanaka, A., Matsuda, M., Okabayashi, T., Pitaksajjakul, P., Rama-
        soota, P., Saito, K., . . . Suzuki, R. (2021). Seroprevalence of
        ﬂavivirus neutralizing antibodies in thailand by high-throughput
        neutralization assay: Endemic circulation of zika virus before 2012.
        <em>mSphere</em>, <em>6</em>(4), 10.1128/msphere.00339-21. Retrieved
        from https://journals.asm.org/doi/abs/10.1128/msphere.00339-21 doi:
        10.1128/msphere.00339-21 1
      </>
    ),
  },
];

const Results = () => {
  return (
    <>
      <MyHead title="Results" />
      <Layout>
        <DocLayout>
          <PaperSection>
            <H1 refId="h1:summary">Summary</H1>
            <P>
              {'"'}STANDUP{'"'} has been developing two high-throughput,
              low-cost, and high-sensitivity testing methods, CFNT and 3D-PCR,
              to address the lack of surveillance for dengue fever. On this
              page, we aim to present the most significant outcomes from the
              development of these testing methods. To understand how these
              results specifically relate to the project, please check the Proof
              of Concept page for more details.
            </P>
          </PaperSection>
          <PaperSection>
            <H1 refId="h1:cfnt-results">CFNT Results</H1>
            <P>
              {'"'}CFNT: Cell Fluorescence Neutralization Test{'"'} is a testing
              method that measures the amount of neutralizing antibodies against
              each serotype of the dengue virus (DENV) present in the
              subject&apos;s serum. In this section, we will present three key
              experimental results that are crucial when implementing CFNT. For
              more details, please see the
              <Link href="/experiments">Experiments</Link>
              page.
              <ol>
                <li>
                  Creation of Infection Detection Cells
                  <ol>
                    <li>Plasmid DNA Construction</li>
                    <li>Transformation into Vero Cells</li>
                  </ol>
                </li>
                <li>Measurement of SRIP&#39;s Infectious Units (IU)</li>
                <li>Room Temperature Cultivation of C6/36</li>
              </ol>
            </P>
            <H2 refId="h2:creation-of-infection-detection-cells">
              1. Creation of Infection Detection Cells
            </H2>
            <P>
              In CFNT, the amount of neutralizing antibodies in the
              subject&apos;s serum is measured by the ratio of fluorescence
              emitted by SRIP&apos;s infection detection cells. Thus, the
              infection detection cells are one of the most critical elements.
              This section discusses the construction of plasmid DNA for
              transformation and the transformation into Vero cells in relation
              to the creation of infection detecting cells.
            </P>
            <H3 refId="h3:plasmid-dna-construction">
              a. Plasmid DNA Construction
            </H3>
            <P>
              The construction of the plasmid DNA was carried out as described
              in the <Link href="/experiments">Experiments</Link> section. A
              synthetic DNA containing genes in the order of loxP, NLS, N-Cre,
              and loxP, mCherry was inserted as an insert into the backbone
              pIRES2-EGFP, and the DNA was amplified in E. coli. The
              electrophoresis photo (<Ref refId="fig:1" />
              A) shows the plasmid extracted from E. coli and the same plasmid
              digested with the restriction enzyme Afl II (1 cutter). From{" "}
              <Ref refId="fig:1" />
              B, it can be seen that the constructed plasmid pCMV N-Cre is 6.9
              kbp, and from the electrophoresis photo
              <Ref refId="fig:1" />
              A, it can be inferred that the linearized pCMV N-Cre digested with
              Afl II is in the appropriate position.
            </P>
            <DocImg
              refId="fig:1"
              src="pages/results/results-fig-1.png"
              alt="1"
              caption={
                <>
                  Analysis by agarose gel electrophoresis about pCMV N-Cre
                  construction
                  <br />
                  <div>
                    A: 0.6% agarose gel image, loaded with extracted pCMV N-Cre
                    from E.coli and pCMV N-Cre digested with Afl II
                  </div>
                  <div>B: pCMV N-Cre plasmid map</div>
                </>
              }
            />
            <H3 refId="h3:transformation-into-vero-cells">
              b. Transformation into Vero Cells
            </H3>
            <P>
              The infection detection cells are created by transforming them
              with pCMV N-Cre, as described in the{" "}
              <Link href="/experiments">Experiments</Link> section. The
              following photo shows the cells emitting green fluorescence before
              being infected with SRIP (<Ref refId="fig:2" />) . For more
              detailed fluorescence images, please refer to the link in the{" "}
              <Link href="/experiments">Experiments</Link> section.
            </P>
            <DocImg
              refId="fig:2"
              src="pages/results/results-fig-2.png"
              alt="2"
              caption={
                <>
                  SRIP infection detecting cells Vero transfected with pCMV
                  N-Cre
                </>
              }
            />
            <P>
              Based on the above results, it can be said that pCMV N-Cre was
              correctly constructed and appropriately transformed. These
              significant findings strongly support the successful creation of
              the infection detection cells.
            </P>
            <H2 refId="h2:measurement-of-srip">
              2. Measurement of SRIP&#39;s Infectious Units (IU)
            </H2>
            <P>
              In CFNT, we use DENV-like SRIP, and as stated in the
              <Link href="/model">Model</Link> section, stable production of
              SRIP is extremely important. To construct a model for calculating
              the amount of SRIP produced, we replicated SRIP [1] produced by
              Dr. Suzuki of the National Institute of Infectious Diseases and
              obtained production data. A series of experiments were conducted
              as described in the
              <Link href="/experiments">Experiments</Link>
              section. The produced SRIP was diluted and used to infect Vero
              cells, and the Infectious Units were measured. Infected cells were
              identified by fluorescent labeling with anti-NS1 antibodies, and
              the number of infected cells in one well was counted (
              <Ref refId="fig:3" />
              ). The results showed that the most SRIP is produced on the second
              day after the plasmid DNA is transformed into HEK293T cells (
              <Ref refId="fig:4" />
              ).
            </P>
            <DocImg
              refId="fig:3"
              src="pages/results/results-fig-3.png"
              alt="3"
              caption={
                <>
                  SRIP-infected Vero cells immunostained green Stained green
                  with an anti-NS1 antibody that binds to NS1 produced by
                  SRIP-infected Vero cells. Cell nuclei were stained blue with
                  DAPI.
                </>
              }
            />
            <DocImg
              refId="fig:4"
              src="pages/results/results-fig-4.png"
              alt="4"
              caption={
                <>
                  Infectious Units of SRIPs according to the number of culturing
                  days
                </>
              }
            />
            <P>
              As a result, we were able to construct an appropriate model for
              calculating the amount of SRIP produced. For more details, please
              see the <Link href="/model">Model</Link> page.
            </P>
            <H2 refId="h2:cultivation-of">
              3. Cultivation of C6/36 cells without an incubator
            </H2>
            <P>
              In CFNT, results are interpreted based on the ratio of
              fluorescence emitted by the infection detection cells. However, as
              stated in the <Link href="/design">Design</Link> section, if we
              can produce infection detection cells using mosquito cells instead
              of mammalian culture cells, which require stringent culture
              conditions, there is a higher possibility that CFNT tests could be
              conducted without an incubator. Therefore, we conducted
              experiments to verify whether C6/36 cells, which are mosquito
              cells, could be cultured without maintaining a 5% carbon dioxide
              concentration (
              <Ref refId="fig:5" />
              ). For more details, please refer to the{" "}
              <Link href="/experiments">Experiments</Link> section.
            </P>
            <P>
              The experiment suggests that culturing is possible without
              maintaining a 5% carbon dioxide concentration if HEPES is added to
              the culture medium. The fact that the growth curve has not fully
              risen is likely because the cells have not yet fully adapted; it
              is thought that if culturing continues under the same conditions
              for a while, the cells may proliferate to the same extent as they
              would under optimal conditions.
            </P>
            <DocImg
              refId="fig:5"
              src="pages/results/results-fig-5.png"
              alt="4"
              caption={
                <>
                  C6/36 cell growth curve C6/36 cells is cultured under optimal
                  conditions (28℃ and 5% CO_2), and not optimal conditons (28℃,
                  0% CO_2 and HEPES 10, 20 or 30 mM)
                </>
              }
            />
            <P>
              Based on these results, it is supported that using C6/36 cells as
              infection detection cells could reduce the equipment costs for
              CFNT.
            </P>
          </PaperSection>
          <PaperSection>
            <H1 refId="h1:functional-avaluation-of-3dpcr">
              Functional evaluation of 3D-PCR
            </H1>
            <P>
              In the experimental stage, we investigated whether the
              conceptualized 3D-PCR actually functions as a technique. The
              experiments were carried out by adding synthetic DNA at random
              positions and operating according to the procedures theorized for
              3D-PCR. Initially, DNA was added to one well, then the number of
              wells was increased; at the current point, the procedure has been
              completed for four different wells.
            </P>
            <P>
              The electrophoresis results shown below represent samples gathered
              from a 3D-PCR experiment in which DNA (pUC19) was added to two
              wells. In this experiment, DNA was added at positions (x,y,z)=(8 ,
              7 , 3) and (4 , 3 , 6) as shown in <Ref refId="fig:6" />. DNA
              amplification can be confirmed at the corresponding coordinates on
              each plane as seen in <Ref refId="fig:7" />. These results prove
              that our conceived 3D-PCR method is functional.
            </P>
            <DocImg
              refId="fig:6"
              src="pages/results/results-fig-6.png"
              alt="4"
              caption={
                <>
                  3D-PCR where (8, 7, 3) and (4, 3, 6) is positive In this case,
                  you can see that Sample x-4, x-8, y-4, y-7, z-3 and z-7 will
                  be positive
                </>
              }
            />
            <DocImg
              refId="fig:7"
              src="pages/results/results-fig-7.png"
              alt="4"
              caption={
                <>
                  Analysis by agarose gel electrophoresis about 3D-PCR where (8,
                  7, 3) and (4, 3, 6) are positive 1.5% agarose gel image, from
                  left to right X coordinate (x-1, x-2, …, x-12, P, N, M), Y
                  coordinate (y-1, y-2, …, y-8, P, N, M), Z coordinate (z-1,
                  z-2, …, z-10, P, N, M). P, N and M means Positive Control,
                  Negative Control and Marker.
                </>
              }
            />
          </PaperSection>
          <CiteRefList citeList={citeList} />
        </DocLayout>
      </Layout>
    </>
  );
};

export default Results;