iGEM 2024 — Team vilnius-lithuania



// @ts-nocheck
import React from "react";
import { Navigation } from "../components/navigation/Navigation";
import { Footer } from "../components/footer/Footer";
import { Header } from "../components/header/Header";
import { Paper } from "../components/paper/Paper";
import { experiments_photo, protocols } from "../static/images";
import { Card, CardBody } from "@material-tailwind/react";

const ExperimentsPage = () => {
  return (
    <>
      <Navigation />
      <Header title="EXPERIMENTS" photo={experiments_photo}></Header>
      <Paper>
        <section className="mb-8 text-justify">
          <h2 className="text-4xl font-bold mb-4" id="protocols">
            Protocols
          </h2>
          <div className="flex justify-center">
            <Card
              className="border border-gray-300 shadow-xl mx-2 bg-gray-50 w-11/12"
              placeholder={undefined}
            >
              <CardBody
                className="p-4"
                style={{
                  height: "600px",
                  overflowY: "scroll",
                  scrollBehavior: "smooth",
                }}
                placeholder={undefined}
              >
                <iframe src={protocols} width="100%" height="550px" />
              </CardBody>
            </Card>
          </div>
          <figcaption className="text-center text-sm mt-2">
            <b>Fig. 1.</b> Protocols
          </figcaption>
        </section>
        <section className="mb-8 text-justify">
          <h2 className="text-4xl font-bold mb-4" id="weekly-lab-work">
            Weekly lab work
          </h2>
          <h2 className="text-2xl font-bold mb-4" id="may">
            May
          </h2>
          <h2 className="text-xl mt-8 font-bold mb-4">May 1-5</h2>
          <p className="py-2">
            Our first week in the lab consisted of revivification and the
            beginning of the cultivation process of our received{" "}
            <em>C.crecestus</em> and <em>H. baltica</em> strains. We also
            conducted a full inventorization of our laboratory.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">May 6-12</h2>
          <p className="py-2">
            This week started with the ending of the bacteria cultivation
            process. After that, we purified their genomic DNA from{" "}
            <em>C. crescentus</em> CB2, CB2A strains, and <em>H. baltica,</em>{" "}
            which we will use in the future. Also, a batch of electrocompetent{" "}
            <em>E. coli </em>Mach1cells was made, and their efficiency was
            tested. First, we attempted to amplify our genes using PCR
            (polymerase chain reaction).
          </p>
          <p className="py-2">
            Preparing stock solutions for our gene knockout experiments. Making
            the necessary LB agar plates for the arriving pCas9 and pCRISPR-SacB
            plasmids. Preparing electrocompetent KRX strain cells.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">May 13-19</h2>
          <p className="py-2">
            We continued our work of amplification. On the side, stocks of
            antibiotics were made, and we will use them to grow overnight mini
            preps and LB agar plates.
          </p>
          <p className="py-2">
            Plated received plasmids (pCasRed plasmid - pCas9, pCRISPR-SacB
            plasmid - pCRISPR, pCas9, and pTargetF (the last two received from
            Songailienė)).
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">May 20-26</h2>
          <p className="py-2">
            The week started with the last PCR reactions being finished. During
            this time, we prepared our vectors (pETDuet and pACYCDuet) for the
            GG (Golden Gate) reaction. By the end of the week, the first
            attempts of GG reactions were made, and the products were
            electroporated into Mach1 electrocompetent cells.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">May 27 - June 2</h2>
          <p className="py-2">
            Along with more gene amplification and GG reactions, we tested the
            validity of our made constructs with cPCR (colony polymerase chain
            reaction) alongside RE (restriction endonuclease) reactions. During
            this week, a new batch of Mach1 electrocompetent cells was tested
            for their efficiency.
          </p>
        </section>

        <section className="mb-8 text-justify">
          <h2 className="text-2xl font-bold mb-4" id="june">
            June
          </h2>
          <h2 className="text-xl mt-8 font-bold mb-4">June 3-9</h2>
          <p className="py-2">
            This week, the work of assembling our plasmids continued, along with
            retrying some of the failed GG reactions and PCR. We successfully
            assembled the CB2 hfsA-hfsB-hfsD plasmid.&nbsp;
          </p>
          <p className="py-2">
            Growing bacteria with plasmids required for gene knock-outs, doing{" "}
            <em>in silico</em> work (primer, homology arm creation), PCR of gDNA
            fragments, and making necessary stock solutions.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">June 10-16</h2>
          <p className="py-2">
            The assembly of plasmids continues. From the already electroporated
            and grown GG products in our Mach1 strain, we did cPCR (colony
            polymerase chain reaction), and after plasmid purification, RE
            reactions were performed. If both tests were successful, we prepared
            and sent the samples for sequencing. This week we successfully
            assembled HB hfsF-hfsC-hfsI plasmid.
          </p>
          <p className="py-2">
            KLD reaction on gDNA plasmids to make them circular. Transformation
            of circular plasmids into Mach1 electrocompetent cells. Performed
            cPCR to check the validity of the plasmid sequence. Did PCR on dDNA
            for wecA gene knockout. Multiplied pCas9 plasmid.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">June 17-23</h2>
          <p className="py-2">
            This week, we kept on assembling and checking our plasmids using the
            methods mentioned above.
          </p>
          <p className="py-2">
            Multiplied pCas9 plasmid and reran dDNA PCRs products through a TBE
            agarose gel. Purified both the pCas9 plasmids and dDNA PCR products.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">June 24-30</h2>
          <p className="py-2">
            The week consisted of plasmid assembly and their sequence
            validation. This week we successfully assembled CB2 hfsF-hfsC-hfsI
            plasmid.
          </p>
          <p className="py-2">
            Redone dDNA PCR from last week because the wrong primers were used.
          </p>
        </section>

        <section className="mb-8 text-justify">
          <h2 className="text-2xl font-bold mb-4" id="july">
            July
          </h2>
          <h2 className="text-xl mt-8 font-bold mb-4">July 1-7</h2>
          <p className="py-2">
            The lab team split into two groups: half of the team kept at it with
            plasmid assembly and sequence validation, and the other half started
            with expression work (multiple tries in KRX strain). So, the job
            consisted of making overnight cultures and a 6 LB medium flask.
            After protein expression, the first SDS-PAGE gels were made and run.
            This week we successfully assembled CB2A hfsA-hfsB-hfsD and CB2A
            hfsF-hfsC-hfsI.
          </p>
          <p className="py-2">
            Performed wecA gene knock-out and prepared gDNAs with dDNAs for
            other gene targets. Stocked up on gene knock-out solutions.
            Electrotransformed gDNAs in Mach1 electrocompetent cells. Prepared
            other required plasmids and sent them for sequencing.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">July 8-14</h2>
          <p className="py-2">
            During this week, some significant changes were made. Our plasmid
            assembly work kept stalling, so we decided to change the vector
            backbone for our cytoplasmic genes. We were using pETDuet-1 from
            this week onwards, we used pRSFDuet-1. Also, constructing the
            plasmids with genes PCR-amplified straight from the genome did not
            work, so we adjusted and, from this week, used 2-step amplification,
            meaning first we amplified the genes from the genome and then used
            the purified PCR product as a matrix for another PCR (overall
            schematic: Genome -&gt; (first PCR) -&gt; Purified firsts PCR
            product -&gt; (second PCR, using first PCR&rsquo;s product as a
            matrix) -&gt; amplified gene for plasmid assembly). We performed
            protein expression (KRX strain) and the corresponding SDS-PAGE gels
            alongside plasmid assembly work. This week we successfully assembled
            CB2 hfsE-hfsJ-hfsG and CB2 hfsL-hfsH-hfsK.&nbsp;
          </p>
          <p className="py-2">
            Prepared gene knock-out solutions and grew KRX electrocompetent
            cells (with pCas9 plasmid). Electrotransforming pTargetF plasmid
            (targeting wecA) and dDNA templates. To validate the sequence PCR
            was performed.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">July 15-21</h2>
          <p className="py-2">
            This week mainly consisted of plasmid assembly and sequence
            validation. On the side, we made a new batch of electrocompetent
            Mach1 cells. This week we successfully assembled CB2A hfsL-hfsH-hfsK
            and HB hfsE-hfsG-hfs.&nbsp;
          </p>
          <p className="py-2">
            Performed experiment to knock out WecF gene. Cured pTargetF and WecA
            from KRX gene with knocked out wecA gene.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">July 22-28</h2>
          <p className="py-2">
            Nothing new happened this week, we kept at it with plasmid
            assemblies and one protein expression (KRX strain) was performed
            with SDS-PAGE gel following right after.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">July 29 - August 4</h2>
          <p className="py-2">
            The first part of the week consisted of finishing the plasmid
            assembly part of our project. Of course, not all the plasmids were
            entirely constructed by this part, but we have the whole system of
            C. crescentus CB2. There is still some work to be done with the CB2A
            strain and <em>H. baltica</em>. From this week on, only one lab team
            member will finish the plasmid assemblies. Also, during this week,
            the grunt work of protein expression (KRX and BL21 (DE3) strains)
            started. This week we successfully assembled HB hfsJ-hfsL-hfsK.
          </p>
        </section>

        <section className="mb-8 text-justify">
          <h2 className="text-2xl font-bold mb-4" id="august">
            August
          </h2>
          <h2 className="text-xl mt-8 font-bold mb-4">August 5-11</h2>
          <p className="py-2">
            The lab's primary human resources were focused on protein
            expressions in different host strains and conditions (BL21(DE3)
            strain). On the side, all the SDS-PAGE gels are being prepared and
            photographed. Also, two new batches of electrocompetent cells were
            made: Mach1 and BL21 (DE3). During all of this, the final plasmid
            assemblies are finished. This week we successfully assembled HB
            hfsA-hfsB-hfsD.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">August 12-18</h2>
          <p className="py-2">
            This week, we continued protein expression (BL21 (DE3) strain) work.
            Our lab lead tried to purify our final product holdfast (the
            protocol was adapted from several other protocols; you can see the
            final version in the protocol book). The product from the
            purification experiment was used for our first WGA blot (the
            protocol is also in the protocol book). During this time, work for
            ori/antibiotic-changed plasmids started. We finished the week by
            preparing our samples and going to ThermoFisher Scientific for
            proteomics. This week we successfully assembled CB2A hfsE-hfsJ-hfsG.
          </p>
          <p className="py-2">
            Made overnight cultures for freshly transformed wecA gDNA
            constructs. We decided to change our strategy for gene knockouts,
            and created new primers for the required constructs.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">August 19-25</h2>
          <p className="py-2">
            We are continuing the work started a week ago for the antibiotic
            switch. Expression (BL21 (DE3) and C41 (DE3) strains) work is still
            continuing. We are narrowing down the optimal conditions and
            determining what host works best. Some of the best results harboring
            expressions were repeated on a larger scale for purification
            experiments.
          </p>
          <p className="py-2">
            Redone gene knock-out. Made electrocompetent BL21 (DE3) cells with
            pCas9 plasmid and purified new dDNA. Made overnight cultures of BL21
            (DE3) + pRED and p708 (flipase) plasmids, also HMS174 + pCas9.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">
            August 26 - September 1
          </h2>
          <p className="py-2">
            During this intense week, a couple of expressions (BL21 (DE3) and
            C41 (DE3) strains) were performed and left overnight to form rings
            with glucose. All the lab's human resources were poured into
            amplifying and running GG reactions with our chosen genes, which
            will be purified using 6X histidine markers and spin columns. Before
            the protein purification process started, we checked the validity of
            every plasmid using already discussed methods. Also, another dot
            blot was performed. We plated our ring formations onto various
            plates (the plates varied in IPTG concentration and antibiotics, for
            in depth information see &ldquo;Engineering&rdquo;). The week ended
            with the first attempts at holdfast purification.
          </p>
          <p className="py-2">
            Purified pRED and p708 plasmids. Made electrocompetent cells (BL21
            (DE3), DH10B, C41 (DE3), Rosetta(DE3)pLysS). Made HMS174 (DE3)
            electrocompetent cells and electrotransforming pRED plasmid into
            them.
          </p>
        </section>

        <section className="mb-8 text-justify">
          <h2 className="text-2xl font-bold mb-4" id="september">
            September
          </h2>
          <h2 className="text-xl mt-8 font-bold mb-4">September 2-8</h2>
          <p className="py-2">
            Starting this week with a new batch of Mach1 electrocompetent cells.
            Expression work started again (Rosetta, BL21 (DE3) and C41 (DE3)
            strains). Another dot blot was prepared with the products obtained
            from last week's holdfast purification experiment. We expressed all
            of our single gene plasmids that were prepared last week. Bioreactor
            tests were executed.
          </p>
          <p className="py-2">
            Performed homology recombineering gene knock-out for DH10B and
            HMS174(DE3). PCR&rsquo;ed the products and checked them on 1% TAE
            agarose gels.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">September 9-15</h2>
          <p className="py-2">
            The last of our expressions (BL21 (DE3) strain) were performed, and
            minor finishing work. Also, a WGA dot blot assay was performed.
          </p>
          <p className="py-2">
            Performed homology recombineering gene knock-out of <em>wecA</em>{" "}
            and <em>wecB</em> genes for <em>E. coli</em> strains DH10B,
            HMS174(DE3), BL21(DE3), Rosetta(DE3)pLysS, C41(DE3). PCR&rsquo;ed
            the products and checked them on 1% TAE agarose gels.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">September 16-22</h2>
          <p className="py-2">
            Expressions (BL21 (DE3) and HMS174 (DE3) strains) and WGA blot were
            performed. A bioreactor experiment was performed. We closed the week
            off with a deep clean of the lab. SEM experiments were done with the
            help of colleagues from the Faculty of Chemistry and Geosciences.
          </p>
          <h2 className="text-xl mt-8 font-bold mb-4">September 23-29</h2>
          <p className="py-2">
            This week we tested our bioreactor. We did FTIR and bright field
            microscopy to analyze holdfast-producing bacteria. Knocked-out
            strains were tested for protein and holdfast production.
          </p>
        </section>
      </Paper>
      <Footer />
    </>
  );
};

export default ExperimentsPage;