iGEM 2023 — Team ui-indonesia


import Hero from '@/components/Hero';
import ReadLayout from '@/components/ReadLayout';
import References from '@/components/Refrences';
import { Heading } from '@/components/TableOfContents';
import { Metadata } from 'next/types';
import React from 'react'

export const metadata: Metadata = {
    title: "Engineering"
}

const headingsData: Heading[] = [
    { text: "Introduction", level: 1, id: "introduction" },
    { text: "Components", level: 1, id: "components" },
    { text: "Salmonella typhimurium A1-R", level: 3, id: "salmonella-typhimurium-a1-r" },
    { text: "Buforin IIb", level: 3, id: "buforin-iib" },
    { text: "6xHis tag", level: 3, id: "6xhis-tag" },
    { text: "p53", level: 3, id: "p53" },
    { text: "SCD derived p53-repressible promoter", level: 3, id: "scd-derived-p53-repressible-promoter" },
    { text: "Pspv2", level: 3, id: "pspv2" },
    { text: "Other genetic components", level: 3, id: "other-genetic-components" },
    { text: "pFPV25", level: 3, id: "pfpv25" },
    { text: "System Overview", level: 1, id: "system-overview" },
    { text: "Assembly", level: 1, id: "assembly" },
    { text: "Future Direction", level: 1, id: "future-direction" },
    { text: "Drug delivery nanoparticles", level: 3, id: "drug-delivery-nanoparticles" },
    { text: "Further attenuation", level: 3, id: "further-attenuation" },
    { text: "Tumor-positive genetic sensor", level: 3, id: "tumor-positive-genetic-sensor" },
    { text: "In-vivo testing", level: 3, id: "in-vivo-testing" },
    {text: "references",level: 2,id: "references"},
];

// Define a TypeScript array of strings
const referenceList: string[] = [
    "Mi Z, Feng ZC, Li C, Yang X, Ma MT, Rong PF. Salmonella-Mediated Cancer Therapy: An Innovative Therapeutic Strategy. J Cancer. 2019;10(20):4765.",
    "Felgner S, Spöring I, Pawar V, Kocijancic D, Preusse M, Falk C, et al. The immunogenic potential of bacterial flagella for Salmonella-mediated tumor therapy. Int J Cancer. 2020;147(2):448–60.",
    "Toso JF, Gill VJ, Hwu P, Marincola FM, Restifo NP, Schwartzentruber DJ, et al. Phase I Study of the Intravenous Administration of Attenuated Salmonella typhimurium to Patients With Metastatic Melanoma. J Clin Oncol Off J Am Soc Clin Oncol. 2002 Jan 1;20(1):142–52.",
    "Zhang Y, Zhang N, Zhao M, Hoffman RM. Comparison of the selective targeting efficacy of Salmonella typhimurium A1-R and VNP20009 on the Lewis lung carcinoma in nude mice. Oncotarget. 2015 Jun 10;6(16):14625–31.",
    "Badie F, Ghandali M, Tabatabaei SA, Safari M, Khorshidi A, Shayestehpour M, et al. Use of Salmonella Bacteria in Cancer Therapy: Direct, Drug Delivery and Combination Approaches. Front Oncol [Internet]. 2021 [cited 2023 Oct 11];11. Available from: https://www.frontiersin.org/articles/10.3389/fonc.2021.624759",
    "Cho JH, Sung BH, Kim SC. Buforins: histone H2A-derived antimicrobial peptides from toad stomach. Biochim Biophys Acta. 2009 Aug;1788(8):1564–9.",
    "Li D, Xu Y. Buforin IIb induced cell cycle arrest in liver cancer. Anim Cells Syst. 2019 Apr 26;23(3):176–83.",
    "Lee HS, Park CB, Kim JM, Jang SA, Park IY, Kim MS, et al. Mechanism of anticancer activity of buforin IIb, a histone H2A-derived peptide. Cancer Lett. 2008 Nov 18;271(1):47–55.",
    "Avrutsky MI, Troy CM. Caspase-9: A Multimodal Therapeutic Target With Diverse Cellular Expression in Human Disease. Front Pharmacol [Internet]. 2021 [cited 2023 Oct 11];12. Available from: https://www.frontiersin.org/articles/10.3389/fphar.2021.701301",
    "Wang Q, Zhu F, Xin Y, Liu J, Luo L, Yin Z. Expression and purification of antimicrobial peptide buforin IIb in Escherichia coli. Biotechnol Lett. 2011 Nov 1;33(11):2121–6.",
    "Spriestersbach A, Kubicek J, Schäfer F, Block H, Maertens B. Chapter One - Purification of His-Tagged Proteins. In: Lorsch JR, editor. Methods in Enzymology [Internet]. Academic Press; 2015 [cited 2023 Oct 11]. p. 1–15. (Laboratory Methods in Enzymology: Protein Part D; vol. 559). Available from: https://www.sciencedirect.com/science/article/pii/S0076687914000688",
    "Zhu G, Pan C, Bei JX, Li B, Liang C, Xu Y, et al. Mutant p53 in Cancer Progression and Targeted Therapies. Front Oncol [Internet]. 2020 [cited 2023 Oct 11];10. Available from: https://www.frontiersin.org/articles/10.3389/fonc.2020.595187",
    "Zhao X, Sun W, Ren Y, Lu Z. Therapeutic potential of p53 reactivation in cervical cancer. Crit Rev Oncol Hematol. 2021 Jan;157:103182.",
    "Nakamura M, Obata T, Daikoku T, Fujiwara H. The Association and Significance of p53 in Gynecologic Cancers: The Potential of Targeted Therapy. Int J Mol Sci. 2019 Nov 4;20(21):5482.",
    "Mircetic J, Dietrich A, Paszkowski-Rogacz M, Krause M, Buchholz F. Development of a genetic sensor that eliminates p53 deficient cells. Nat Commun. 2017 Nov 13;8(1):1463.",
    "Valdivia RH, Falkow S. Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction. Mol Microbiol. 1996 Oct;22(2):367–78.",
    "Subramanian K, Payne B, Feyertag F, Alvarez-Ponce D. The Codon Statistics Database: A Database of Codon Usage Bias. Mol Biol Evol. 2022 Aug 1;39(8):msac157.",
    "Mirgayazova R, Khadiullina R, Chasov V, Mingaleeva R, Miftakhova R, Rizvanov A, et al. Therapeutic Editing of the TP53 Gene: Is CRISPR/Cas9 an Option? Genes. 2020 Jun 25;11(6):704.",
    "Chen W, Zhu Y, Zhang Z, Sun X. Advances in Salmonella Typhimurium-based drug delivery system for cancer therapy. Adv Drug Deliv Rev. 2022 Jun 1;185:114295."
  ];

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            <Hero title='Engineering' />
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                <section className='mt-4'>
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                        <h2 id="introduction" className="mt-8 text-4xl scroll-mt-20">Introduction</h2><br />
                        <p>In essence, our system augments the innate anticancer properties of Salmonella.
                            These properties rely on immunogenicity, but wild-type Salmonella remain too pathogenic
                            to be reliably used therapeutically as is. Attenuations to pathogenicity potentially also
                            reduce its anticancer efficacy, though not always and either have no effect towards or amplifying it.
                            (1) Therefore, a balance needs to be struck between the strength of the immune system wrought upon the
                            cancerous environment and the host. Many research teams have tried balancing these aspects and some even
                            made it to clinical testing. Others find exceptions to the dichotomy. (2) Our team decided to sidestep this
                            balancing act entirely by introducing an external element to Salmonella. In other words, we are adding an
                            extra warhead to existing guided missiles</p><br />
                        <p>Our system augments an attenuated Salmonella strain with expression and release of
                            anticancer peptides. We impart this ability by inserting a gene expressing anticancer peptide under
                            a promoter that is preferentially active inside our target cells (cervical cancer cells).</p>
                    </div>
                </section>

                <section className='mt-4'>
                    <h1 id="components" className="mt-8 text-4xl scroll-mt-20">Components</h1>
                    <div className="rounded-xl bg-base-content text-black p-4 shadow-[0_3px_10px_rgb(0,0,0,0.2)] flex flex-col gap-6">
                        <h2 id="salmonella-typhimurium-a1-r" className="mt-8 text-4xl scroll-mt-20">Salmonella typhimurium A1-R</h2>

                        <p>
                            Attenuated Salmonella strains are currently being explored as either cancer adjuvant or monotherapy. Anticancer properties of Salmonella are mostly immune-mediated. It essentially increases the “signal” coming from the tumor microenvironment by adding its own antigens and other immunostimulatory signals to the cancer cells after colonizing it. The end-result is enhanced immune system recruitment in the microenvironment hence augmented cancer cell death. At a more local level, it also directly competes for resources with the cancer cells. (1) Attenuation is necessary as wild-type Salmonella is too pathogenic such that the strength of the immune system wrought upon the cancer cells has too much collateral on the host. Attenuation seeks to limit this collateral and, ideally, eliminate it entirely. (2
                        </p>
                        <p>
                            Data from a phase 1 clinical trial (3) utilizing intravenous administration of attenuated Salmonella revealed several potential pitfalls. It would seem that colonization of the tumor microenvironment is not as potent as demonstrated in murine cells. Mechanisms underlying this difference have yet to be explored. However, once Salmonella colonizes the tumor cells, elimination becomes minimal, but not enough Salmonella colonizes the tumor microenvironment to provide ample anticancer properties. In other words, not enough missiles reach the target to ensure satisfactory destruction. Hence, our design adds additional anticancer properties through anticancer peptide release. One area in which VNP20009 did not fail, however, was safety. Intravenous infusion was remarkably tolerated by trial patients. Fever, hypotension, thrombocytopenia, anemia, persistent bacteremia, low WBC count (only observed at lower dose levels), hypophosphatemia, hyperbilirubinemia, diarrhea, vomiting, nausea, and elevated alkaline phosphatase and transaminases were observed, but none worsened clinical outcomes of the patients nor severely complicated the patients’ conditions.
                        </p>
                        <p>Initially, we planned to utilize VNP20009, the strain used in the only phase 1 clinical trial. However, upon further analysis of alternative attenuated strains, we concluded that A1-R is the best option. As mentioned, VNP20009 does not demonstrate efficacious anticancer activity. So following our initial design philosophy of increasing payload, we found that A1-R was more potent than VPN20009. (4) The same study also found that A1-R in-vivo safety was superior. Considering the acceptable safety of VNP20009 in human subjects, we assessed A1-R as even a safer option. With these considerations, A1-R was decided to be the strain of choice for this system.
                        </p>

                        <h2 id="buforin-iib" className="mt-8 text-4xl scroll-mt-20">Buforin IIb</h2>

                        <p>
                            Bacterial-mediated cancer treatment (BMCT) is a quite recent development in the search for a cure for cancer. It uses different attenuated bacteria to target tumors and exert various antitumour effects. Among the many bacteria used in BMCT, one of them is Salmonella enterica servoar Typhimurium. This bacteria species is capable of targeting and colonizing tumors. It can be used to directly kill tumor cells or deliver anti-tumor agents. (1) The method that our team used is using S. typhimurium to deliver anti-tumor agents, more specifically Buforin IIb
                        </p>
                        <figure className='flex flex-col items-center my-6'>
                            <img src="https://static.igem.wiki/teams/4749/wiki/dry-lab/buforin-1.png" alt="" width={500} height={500} />
                            <figcaption className='text-sm italic  md:w-1/2 text-center'>Antitumor mechanism of S. typhimurium. (5)
                            </figcaption>
                        </figure>

                        <p>
                            Buforin IIb is an antimicrobial peptide with a helix-hinge-helix structure derived from histone 2A which was isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans.(6) This peptide contains 21 amino acids and exhibits a strong antimicrobial activity against a variety of microorganisms. It has a unique mechanism of action such as rapidly crossing bacterial membranes without lysing the cells. It kills bacteria by interacting with intracellular macromolecules. (7)                        </p>
                        <figure className='flex flex-col items-center my-6'>
                            <img src="https://static.igem.wiki/teams/4749/wiki/dry-lab/buforin-2.png" alt="" width={500} height={500} />
                            <figcaption className='text-sm italic  md:w-1/2 text-center'>Ribbon model of Buforin IIb. (6)
                            </figcaption>
                        </figure>
                        <p>
                            Buforin IIb is cytotoxic to cancer cells, but not to normal proliferating cells thus making it safer to use in a cancer treatment. Buforin also requires gangliosides on cell surface for internalization, something a cancer cell is abundant of, thus making it incredibly selective. Buforin IIb induces apoptosis to cancer cells by activating pro-caspase 9 and pro-caspase 3 which initiates mitochondria-dependent apoptosis, thus suppressing the progression of cancer. (8)
                        </p>
                        <figure className='flex flex-col items-center my-6'>
                            <img src="https://static.igem.wiki/teams/4749/wiki/dry-lab/buforin-3.png" alt="" width={500} height={500} />
                            <figcaption className='text-sm italic  md:w-1/2 text-center'>Mechanism of Cell Death. (9)
                            </figcaption>
                        </figure>
                        <p>
                            We had several other anticancer peptide candidates (Chrysophsin-1,2,3, UM-6, and HPRP-A1-TAT). Buforin IIb was singled out as it was the only peptide with thorough documentation on its cell specificity. It is documented to selectively act in negatively charged cells, a common property of cancer cells. This means that the peptides will not be wasted on healthy cells and, subsequently, healthy cells will not suffer destruction. This specificity increases the safety profile of our system as Salmonella already activates a systemic response even when attenuated. (8,10) In addition, buforin IIb is also more well-documented in literature than the other candidates as well. This abundance will assist the team in troubleshooting any problems that might arise.
                        </p>

                        <h2 id="6xhis-tag" className="mt-8 text-4xl scroll-mt-20">6xHistag</h2>
                        <p>
                            A polyhistidine tag, commonly referred to as a His tag, is a collection of several histidine molecules used as a "tag" or marker that is attached to a protein. Histidine has a natural tendency to chelate with metal ions, making it easily bind to metals like nickel. In the case of recombinant proteins, the His tag is introduced into the protein through genetic engineering, typically consisting of two hexahistidine motifs. The purification of His-tagged proteins is achieved by exploiting their strong affinity for metal ions, a process known as affinity chromatography. Ni-NTA (Nickel-nitrilotriacetic acid) is often employed for this purpose, as it relies solely on the chelating site and is thus compatible with both the native and denatured protein structures. Following binding to the metal ions, the protein is subsequently washed with a specific solution, which may include compounds such as imidazole, EDTA, or conditions with a pH range of 5.9 to 4.5. This purification process ensures the isolation of the target protein with the His tag intact, allowing for further research or applications in various fields of study. (11)

                        </p>

                        <h2 id="p53" className="mt-8 text-4xl scroll-mt-20">p53</h2>
                        <p>
                            If the body is a complex, self-regulating machine that performs quality assurance checks every single time on every part of itself, then cancer cells are the “bug” of the software that enables certain parts to be detached and work independently, stealing the resources meant for the surrounding parts and disrupting the whole mechanism. Early signs of cancer cells are continuously checked, detected, and destroyed in an unending process of regulation.
                        </p>
                        <p>
                            The main agent that suppresses cancer cells from being “born” is the p53 gene, (12) the principal  cellular responder to various stress signals such as oncogene activation, DNA damage, hypoxia, reactive oxygen species, and many more. Upon activation, tumor suppressor p53 will elicit a lot of cellular responses that induce arrested cell cycle, senescence, apoptosis, and even ferroptosis
                        </p>
                        <p>
                            Mutation of p53 gene in a cell is crucial to the formation of cancer cells, being the most commonly mutated gene in human cancers. (12) Mutated p53 leads to unexpressed self-destruction sequence, allowing cancer cells to live and proliferate. As such, p53 mutation is an integral part in cancer cell detection of the recent era. This ubiquity also extends to our cancer of choice. Cervical cancer cells have shown to reliably have mutated p53 proteins as a key part of their pathophysiology. (13,14) Therefore, p53 is a viable marker to detect cervical cancer cells.
                        </p>
                        <p>
                            In this project, we exploit the tendency of cancer cells to express mutated p53 as a genetic marker to denote the tumor microenvironment. A genetic sensor detecting mutated p53 is the basis of our system to selectively release our payload augmentation in an environment in which it can be most efficacious.
                        </p>

                        <h2 id="scd-derived-p53-repressible-promoter" className="mt-8 text-4xl scroll-mt-20">SCD derived p53-repressible promoter</h2>
                        <p>
                            Prior testing by Mircetic J et al (15) revealed genetic elements sensitive towards wild-type p53 derived from p53 regulated genes. The elements were sensitive not only to the absence or presence of p53, but could also differentiate between wild-type and mutated p53. We decided to use an SCD-derived element as it is wild-type p53 repressible and does not repress downstream expression when exposed to mutant p53. Additionally, this sensor has the impressive ability to respond to an environment expressing both wild-type and mutated p53 by repressing downstream genes. This sensor has also been proven to function in-vivo as well. Effectively, this becomes the sensor to trigger the detonation of our additional warhead. Subsequently, we would like to also introduce this promoter into iGEM parts database.

                        </p>

                        <h2 id="pspv2" className="mt-8 text-4xl scroll-mt-20">Pspv2</h2>
                        <p>
                            Pspv2 <a href='https://parts.igem.org/wiki/index.php?title=Part:BBa_K112706' className=' link text-accent'>(Part:BBa K112706)</a> is a constitutive promoter compatible in Salmonella. It will be used to constitutively express wild-type p53. Wild-type p53 will be how our system recognises a “neutral” environment i.e. not a cancer cell and keeps expression of buforin IIb off. Competitive inhibition of wild-type p53 by mutated p53 once Salmonella colonizes the cancer cells will alleviate the repression and allow free expression of buforin IIb.

                        </p>

                        <h2 id="other-genetic-components" className="mt-8 text-4xl scroll-mt-20">Other genetic components</h2>
                        <p>
                            The other genetic components of the system were taken from iGEM parts database. These components are:
                        </p>
                        <ul className="list-disc pl-6 space-y-2">
                            <li>
                                Spacer 02 <a className="font-bold link text-accent">(Part: BBa K3831026):</a>
                            </li>
                            <li>
                                Spacer_7 <a className="font-bold link text-accent">(Part: BBa K3831015):</a>
                            </li>
                            <li>
                                Double terminator (B0010-B0012) <a className="font-bold link text-accent">(Part: BBa B0015):</a>
                            </li>
                            <li>
                                RBS by Elowitz <a className="font-bold link text-accent">(Part: BBa B0034):</a>
                            </li>
                        </ul>

                        <p>
                            Among the available spacers in the iGEM parts registry, Spacer 02 and Spacer_7 were chosen because they are the shortest and have the lowest GC content. Length and GC content were a concern brought by the gene synthesis process (Twist Bioscience) Our initial designs used a wider variety of spacers which resulted in genes that are incompatible for synthesis. Therefore, spacer selection was tweaked as it was the only genetic element easily swappable to meet the GC and length criteria. Meanwhile, the double terminator was selected for reliability and RBS for expression strength
                        </p>
                        <p>
                            We also utilized a rigid spacer for 6xHis tags not found in the iGEM parts database. The spacer functions to ensure that the tags do not interfere or to minimize any interference towards protein folding. Simulated protein folding yielded inconclusive results, therefore we chose to err on the side of caution and include the spacer. Subsequently, we would like to add this spacer to the iGEM parts registry.
                        </p>

                        <h2 id="pfpv25" className="mt-8 text-4xl scroll-mt-20">pFPV25</h2>
                        <p>
                            pFPV25 was a gift from Raphael Valdivia (Addgene plasmid # 20667 ; <a href="http://n2t.net/addgene:20667" className='link text-accent'>http://n2t.net/addgene:20667</a> ; RRID:Addgene_20667). It is one of few commercially available expression vectors tested with Salmonella typhimurium with ample documentation and performs well in acidic conditions which lines up with our target tumor microenvironment. (16)
                        </p>
                    </div>
                </section>

                <section className='mt-4'>
                    <div className="rounded-xl bg-base-content text-black p-4 shadow-[0_3px_10px_rgb(0,0,0,0.2)]">
                        <h2 id="system-overview" className="mt-8 text-4xl scroll-mt-20">System Overview</h2><br />
                        <p>
                            Expression of wild-type p53 from plasmid B will repress expression of buforin IIb by plasmid A.
                            After tumor colonization, mutated p53 expressed by cancer cells competitively inhibits wild-type p53
                            and relieves repression of the p53 repressible promoter. Buforin IIb will start to be expressed and
                            accumulate inside Salmonella which have colonized cancer cells. As buforin IIb also has antimicrobial
                            activity, once sufficient concentrations have accumulated, Salmonella will lyse and release the peptide
                            inside the tumor cells. Then, buforin IIb will act as an anticancer peptide and augment the immune-mediated
                            destruction of cancer cells stimulated by Salmonella alone.

                        </p><br />
                        <p>In other words, our system can be summarized into these steps:
                        </p><br />
                        <ul className="list-disc pl-6 space-y-2">
                            <li>Modified Salmonella is injected into the patient.</li>
                            <li>Salmonella targets cervical cancer cells and is phagocytosed.</li>
                            <li>A genetic sensor detects the cancer microenvironment.</li>
                            <li>Expression of buforin IIb is induced.</li>
                            <li>A critical mass of buforin IIb accumulates inside Salmonella and induces autolysis.</li>
                            <li>Buforin IIb is released into cancer cells.</li>
                            <li>Buforin IIb kills cancer cells.</li>
                        </ul>
                    </div>
                </section>

                <section className='mt-4'>
                    <div className="rounded-xl bg-base-content text-black p-4 shadow-[0_3px_10px_rgb(0,0,0,0.2)]">
                        <h2 id="assembly" className="mt-8 text-4xl scroll-mt-20">Assembly</h2><br />
                        <p>
                            The following tables describe the gene insert assemblies to be transformed into Salmonella
                            for our system. Gene inserts are flanked by restriction sites for EcoRI on 5’ and  HindIII on 3’.
                            These restriction sites were chosen as other restriction sites available on pFPV25 are also present
                            within the gene inserts. Codon optimization was also conducted manually referencing Codon Statistics Database.
                            (17)
                        </p>
                        <figure className='flex flex-col items-center my-6'>
                            <img src="https://static.igem.wiki/teams/4749/wiki/dry-lab/dry-tabel-1.png" alt="" />
                            <figcaption className='text-sm italic  md:w-1/2 text-center'>Gene inserts construction.

                            </figcaption>
                        </figure>
                        <figure className='flex flex-col items-center my-6'>
                            <img src="https://static.igem.wiki/teams/4749/wiki/dry-lab/dry-tabel-2.png" alt="" />
                            <figcaption className='text-sm italic  md:w-1/2 text-center'>Gene inserts nucleotide sequences.

                            </figcaption>
                        </figure>
                        <figure className='flex flex-col items-center my-6'>
                            <img src="https://static.igem.wiki/teams/4749/wiki/dry-lab/dry-tabel-3.png" alt="" />
                            <figcaption className='text-sm italic  md:w-1/2 text-center'>Genetic elements nucleotides sequences.

                            </figcaption>
                        </figure>
                    </div>
                </section>

                <section className='mt-4'>
                    <h2 id="future-direction" className="mt-8 text-4xl scroll-mt-20">Future Direction</h2>
                    <div className="rounded-xl bg-base-content text-black p-4 shadow-[0_3px_10px_rgb(0,0,0,0.2)] flex flex-col gap-6">
                        <h2 id="drug-delivery-nanoparticles" className="mt-8 text-4xl scroll-mt-20">Drug delivery nanoparticles</h2>
                        <figure className='flex flex-col items-center my-6'>
                            <img src="https://static.igem.wiki/teams/4749/wiki/dry-lab/future-1.png" alt="" width={500} height={500} />
                            <figcaption className='text-sm italic  md:w-1/2 text-center'>Mechanism of Cell Death. (9)
                            </figcaption>
                        </figure>
                        <p>
                            Salmonella Typhimurium, a well-studied engineered facultative anaerobe, has been extensively modified for various purposes, including serving as carriers for oral vaccine delivery. This approach offers significant advantages over traditional intravenous vaccination methods, as it is not only cost-effective but also less toxic, also explored the use of synthetic polymer-coated live bacteria to target and damage cancer cells, incorporating multiple functionalities to enhance tumor-targeting efficiency, as demonstrated by Xiang et al. Moreover, the concept of "microbotics" has emerged, wherein bacteria are employed as vehicles for delivering nanoparticles and cargo. This approach leverages the invasive properties of bacteria for the delivery of nucleic acids and other payloads. Building on these previous studies, our work aims to coat Salmonella with nanoparticles formed from cationic polymers and DNA. This innovative approach is designed to overcome multiple challenges associated with oral DNA vaccine delivery. Cationic polymers have proven to be promising non-viral gene delivery vectors, as they can spontaneously form nanoscale polymer/DNA complexes (polyplexes) through electrostatic interactions. One notable advantage of cationic polymers is their strong buffering capacity, which enables them to escape from endo/lysosomes within cells through a phenomenon known as the "proton sponge" effect. Calcium carbonate (CaCO3) nanoparticles will serve as carriers for drug delivery. When these nanoparticles are administered orally and enter the body, they will disperse and eventually reach the target cancer cells. Subsequently, the drug carried by the nanoparticles will be released, entering the nucleus of the cancer cells and effectively treating the cancer. (19)
                        </p>

                        <h2 id="further-attenuation" className="mt-8 text-4xl scroll-mt-20">Further attenuation</h2>
                        <p>
                            As previously enumerated, A1-R is an excellent Salmonella strain for use in cancer therapeutics. It is widely studied and therefore its properties well-documented. However, it is also true that our team did not fully explore further modifications to the A1-R strain to create novel strains which have even better and tailored functions for our purposes. For example, knockout of inner membrane ring and basal body component FliF or the flagellum-specific ATPase complex FliHIJ of the flagellar machinery in Salmonella provides better efficacy in treating cancer cells, despite a loss of flagella intuitively meaning attenuated ability for colonization and reduced immune response. (2) Furthermore, the issue of rapid intravascular clearance found in the phase 1 clinical trial (3) was sidestepped. If investigated and proper modifications are made to solve this issue, our increased payload along with more thorough colonization increases efficacy even further. In conclusion, given the opportunity, further modifications to the A1-R strain should be investigated to increase its efficacy as an anticancer drug.

                        </p>

                        <h2 id="tumor-positive-genetic-sensor" className="mt-8 text-4xl scroll-mt-20">Tumor-positive genetic sensor</h2>
                        <p>
                            Our current genetic sensor relies on a change of substance instead of absolute detection of a marker. While the study on which we base our genetic sensor designed the p53 responsive elements as a method of detecting p53 deficient or mutant cells, the genetic sensor effectively detects wild-type p53 and its absence instead of mutant p53. This is why our design relies on competitive inhibition of the sensor with mutant p53 to function. This introduces an additional failure point which has to be taken into account when troubleshooting results. If a genetic sensor exists which activates directly on mutant p53 or another cancer cell genetic marker is found with a genetic sensor that detects it directly, it would make for a handsome replacement of our genetic sensor granted it actually experimentally succeeds.
                        </p>

                        <h2 id="in-vivo-testing" className="mt-8 text-4xl scroll-mt-20">In-vivo testing</h2>
                        <p>
                            A fairly obvious avenue left to be explored is in-vivo testing. Despite extrapolating safety profiles, it remains that our chosen strain has not been human tested and our system has no safety experiments. It is essential that the safety of A1-R expressing buforin IIb through our system is assessed in-vivo that it may provide a baseline expectation for human tests.
                        </p>
                    </div>
                </section>

                <References referenceList={referenceList}/>
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import Hero from '@/components/Hero'
import ReadLayout from '@/components/ReadLayout'
import React from 'react'

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const imageUrls:string[] = [
  prefix + 'a-nitrasi-guanidin-hidroklorat.jpg',
  prefix + 'b-quenching.jpg',
  prefix + 'c-pemurnian-ngu.jpg',
  prefix + 'd-sintesis-mng.jpg',
  prefix + 'e-hasil-murni-mng.jpg',
  prefix + 'f-produk-akhir.jpeg',
];
const imageUrls2:string[] = [
  prefix2 + '20231010-130928.jpg',
  prefix2 + '20231015-101356.jpg',
  prefix2 + 'Salmonella-Thypimurium-ATCC_14028_di_medium_SS_Agar.jpg',
  prefix2 + 'Streak_Salmonella_ke_medium_agar_SS.jpg',
  prefix2 +'Transformasi_salmonella_kultur_A.jpg',
];

export default function page() {
  return (
    <>
      <Hero title={'Experiment'} />
      <ReadLayout headingsData={[]}>
        <div className='flex flex-col gap-4'>
          <Experiment images={imageUrls} id={''} title={'MNNG Synthesis'}/>
          <Experiment images={imageUrls2} id={''} title={'Salmonella A1-R prodcution'}/>
        </div>
      </ReadLayout>

    </>
  )
}