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@LNJC
LNJC authored Oct 11, 2023
2 parents 8822bdc + f8adb7f commit c5c28b0
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43 changes: 43 additions & 0 deletions gatsby-config.mjs
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Expand Up @@ -72,6 +72,46 @@ const config = {
path: "./src/pages/",
},
},
{
resolve: "gatsby-plugin-remote-images",
options: {
nodeType: "TeamMemberYaml",
imagePath: "picturePath",
name: "dynamicImage",
// siteMetdata could be undefined, but is not in our use case, so use ! (definitely assigned)
prepareUrl: url => `${config.siteMetadata.assetBasePath}${url}`,
},
},
{
resolve: "gatsby-plugin-remote-images",
options: {
nodeType: "SponsorYaml",
imagePath: "logoPath",
name: "dynamicImage",
// siteMetdata could be undefined, but is not in our use case, so use ! (definitely assigned)
prepareUrl: url => `${config.siteMetadata.assetBasePath}${url}`,
},
},
{
resolve: "gatsby-plugin-remote-images",
options: {
nodeType: "ProminentLogoYaml",
imagePath: "logoPath",
name: "dynamicImage",
// siteMetdata could be undefined, but is not in our use case, so use ! (definitely assigned)
prepareUrl: url => `${config.siteMetadata.assetBasePath}${url}`,
},
},
{
resolve: "gatsby-plugin-remote-images",
options: {
nodeType: "HomepageCardYaml",
imagePath: "picturePath",
name: "dynamicImage",
// siteMetdata could be undefined, but is not in our use case, so use ! (definitely assigned)
prepareUrl: url => `${config.siteMetadata.assetBasePath}${url}`,
},
},
{
resolve: "gatsby-plugin-manifest",
options: {
Expand Down Expand Up @@ -165,6 +205,9 @@ const config = {
},
},
`@genoswitch/gatsby-transformer-gitinfo`,
`gatsby-plugin-image`,
`gatsby-plugin-sharp`,
`gatsby-transformer-sharp`, // Needed for dynamic images
`gatsby-plugin-no-sourcemaps`,
{
resolve: `gatsby-plugin-mdx`,
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13 changes: 8 additions & 5 deletions package.json
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Expand Up @@ -21,9 +21,9 @@
"dependencies": {
"@emotion/react": "^11.11.1",
"@emotion/styled": "^11.11.0",
"@mui/icons-material": "^5.14.12",
"@mui/material": "^5.14.12",
"gatsby": "^5.12.5",
"@mui/icons-material": "^5.14.13",
"@mui/material": "^5.14.13",
"gatsby": "^5.12.6",
"html-react-parser": "^4.2.2",
"immutability-helper": "^3.1.1",
"katex": "^0.16.9",
Expand All @@ -42,15 +42,18 @@
"@types/react-image-gallery": "^1.2.1",
"@types/webpack-stats-plugin": "^0.3.3",
"gatsby-plugin-canonical-urls": "^5.12.0",
"gatsby-plugin-image": "^3.12.1",
"gatsby-plugin-manifest": "^5.12.1",
"gatsby-plugin-mdx": "^5.12.0",
"gatsby-plugin-mdx": "^5.12.1",
"gatsby-plugin-no-sourcemaps": "^5.12.0",
"gatsby-plugin-pnpm": "^1.2.10",
"gatsby-plugin-postcss": "^6.12.0",
"gatsby-plugin-preact": "^7.12.0",
"gatsby-plugin-sitemap": "^6.12.0",
"gatsby-plugin-remote-images": "^3.6.6",
"gatsby-plugin-sitemap": "^6.12.1",
"gatsby-source-filesystem": "^5.12.0",
"gatsby-transformer-genetic-sequences": "^1.1.0",
"gatsby-transformer-sharp": "^5.12.1",
"gatsby-transformer-yaml": "^5.12.0",
"gatsby-transformer-yaml-full": "^5.0.0",
"gatsby-yaml-full-markdown": "^5.0.1",
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42 changes: 41 additions & 1 deletion pages/mdx/biology.mdx
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Expand Up @@ -136,7 +136,47 @@ Hardware page</a>.

To increase the accessibility of our test, we have chosen to use miRPA to amplify the miRNA taken in
our sample. This allows the miRNA to be isothermally amplified, and therefore doesn’t require the use
of an expensive thermocycler. More information on the probes used can be found on our <a href="#" onClick={() => navigate("/software/")}>Software page</a>.<h2 style={{ paddingTop: 8 }}>MicroRNA</h2>
of an expensive thermocycler. More information on the probes used can be found on our <a href="#" onClick={() => navigate("/software/")}>Software page</a>.

<h3 style={{ paddingTop: 4 }}>Our Three-Step Process </h3>
<h4 style={{ paddingTop: 4 }}>Step 1: Extraction of miRNA </h4>
Extraction of miRNA is done using electromagnetic particles.
<img
style={{ maxWidth: "100%" }}
src="https://static.igem.wiki/teams/4642/wiki/biology/step1.webp"
/>
These are the steps:
Samples of blood are taken from a patient and added to a solution containing magnetic nanoparticles. These nanoparticles often consist of a metal (usually iron) oxide and are also coated with a biotin-streptavidin bonded anti-miRNA strand. A set of electromagnets is turned on and off in quick succession, causing the nanoparticles to move and stir the solution. During this process, target miRNAs bond to the complementary anti-miRNA via base-pairing. Afterwards, the magnetic beads are pulled to the side of the container while the rest of the solution is removed. The miRNA is removed from the beads using an elution buffer. The previous steps are repeated three times in order to remove the maximum possible amount of miRNA for amplification.

<h4 style={{ paddingTop: 4 }}>Step 2: miRPA </h4>
As seen in RIBOTOX, it is difficult to discriminate between leaky expression of toehold switches and {"<"}2.25M concentrations of miRNAs, and there is no data giving concentrations of the trigger miRNAs we are concerned with online.

Therefore, to ensure miRNA concentration is high enough for them to be detected by the toehold switches, we need to amplify our microRNAs. Not only is PCR difficult to perform on microRNAs due to their short length, we need to find a solution that will allow amplification to take place isothermally, in a single tube, increasing the accessibility of our tests.

Recombinase Polymerase Amplification (RPA) is a single tube, isothermal alternative to PCR, which can amplify dsDNA strands. So, before RPA can work, we need to reverse transcribe miRNA into DNA. This can be done using miRPA.

Two DNA probes, one with 5’ phosphorylation, bind to the miRNA, and are ligated together by DNA ligase. Then, primers are added, with DNA polymerase, and complementary strands to the ligated probes are synthesised. Then, RPA can take place: primers, which are associated with recombinase protein dislodge the strands, replicating them in a similar method to PCR, but as no heat cycles are required to break up the strands, the process can take place isothermally.

In order for the miRNA to be detected, we use ‘asymmetric RPA’: an excess of forward primers are added (usually 5x the amount), so an excess of the strand that was originally miRNA form is produced, so there is now ssDNA with the miRNA code in DNA. This can be detected by toehold switches. In order to design probes for miRPA, we can use NUPACK’s design functions in its API to find probes which can bind to the miRNAs, but have overhangs which do not bind within themselves, to ensure primers can easily anneal to them.
<img
style={{ maxWidth: "100%" }}
src="https://static.igem.wiki/teams/4642/wiki/biology/step2.webp"
/>

<h4 style={{ paddingTop: 4 }}>Step 3: Charaterisation of toehold switches </h4>

To detect this now amplified miRNA, we made use of toehold switches. Conventionally, toehold switches open in response to the presence of a specific trigger, usually a single miRNA strand, however this can create issues regarding specificity due to how many miRNA several different conditions can share. Our solution is to use AND gates made of RNA to detect a combination of miRNA all specific to 1 disease, solving this specificity problem.

The RNA and gates are specially designed to join miRNA strands for a single disease together and create a trigger complex, the switches are designed to have a binding site that is complementary to the trigger complex’ unpaired bases. When the trigger complex binds to the switch, the switch collapses, exposing the RBS and start codon allowing for the translation of a reporter protein.

As seen earlier, our switches are multiplexed allowing us to detect 4 diseases at once. Each switch has their own binding site and reporter protein depending on the condition the switch is designed to detect.
<img
style={{ maxWidth: "100%" }}
src="https://static.igem.wiki/teams/4642/wiki/biology/step3.webp"
/>


<h2 style={{ paddingTop: 8 }}>MicroRNA</h2>
Our project uses microRNA biomarkers to indicate the presence of disease. MicroRNAs (miRNAs) are
small, highly conserved non-coding RNA molecules involved in the regulation of gene expression.<Reference number={1}/>

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26 changes: 1 addition & 25 deletions pages/mdx/description.mdx
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Expand Up @@ -8,30 +8,6 @@ import { Divider, List } from "@mui/material";

# Project Description

Project GENOSWITCH’s primary aim is to develop a genetic engineering-based cell-free modular test for microRNA (miRNA) biomarkers that can detect cases simultaneously much sooner than current diagnostic methods, so medication can be taken and patients’ symptoms can be tracked by their doctor.

The secondary aim was to create a framework with which researchers and iGEM teams in the future can develop tests for multiple conditions at low miRNA concentrations, and also to develop a comprehensible laymen’s software tool to increase the use of in-silico experimentation, saving costs in the lab.

The final goal was to spread awareness about women’s health and the specific conditions that are associated with it, which, despite their prevalence, are relatively unknown, and too often damages lives in both the developed and less developed worlds.

<List>
<Divider variant="inset" component="li" />
</List>

We will illustrate our modular mechanism using 4 conditions in Obstetrics and Gynaecology, whereby the relevant free-floating miRNAs anneal to the genetically modified composite circuits we have ligated in K12 E. Coli strain.

Each input will correspond to a unique reporter fluorescent protein – namely GFP, mCherry, mCerulean and Venus.

The light expressed would be quantifiably measured using a luminometer - displaying a diagnosis result.

Our mechanism is applicable to all conditions with upregulated sncRNAs or other blood biomarkers in the form of nucleic acids - this is what makes our project modular.

We also have created a simple software tool that will enable potential users to design the relevant composite circuit in-silico without the need for complex coding or thermodynamic calculations.

<List>
<Divider variant="inset" component="li" />
</List>

This year, the focus of our project is on eradicating the outdated screening procedures now used for illnesses affecting women's health. Our approach uses multiplexed regulation, a synthetic amalgamation circuit with four toehold switches for each of the four conditions (PCOS, Endometriosis, Breast Cancer, and Ovarian Cancer), which each test for three microRNAs. The switches have genetic 'AND' gates, which provide high specificity. With the use of the collaborative database we have begun, our research is unique in that it permits the miRNA binding sites to be switched around.

One blood sample can be used to test for many miRNAs simultaneously, and the findings are available much faster than with earlier techniques. Given the presence of the correct miRNA, the toehold switches will produce various fluorescent proteins once they have unfolded. The chosen fluorescent proteins can be evaluated independently because they have different wavelengths.
Expand All @@ -48,4 +24,4 @@ It is being shipped via appimages and ships with a beta version of NUPACK that w

The final objective was to raise awareness about women's health and the specific illnesses that are related to it. These conditions are largely unknown while being common and frequently have a negative impact on lives in both developed and developing countries. We have carefully considered how our sensor might be applied in a clinical environment as a component of our work in human practises. As part of our integrated human practises, we spoke with numerous clinicians and established a plan for how our test might be utilised in screening programmes. On their advice, we have also increased our sensor's sensitivity and specificity. We have also looked into the potential detection of other diseases by our sensor and the advantages of our approach.

In a clinical implementation, miRNAs extracted from blood serum would be added to a cell free system containing an excess of our amalgamation. The unique toehold switches would detect specific miRNAs and regulate translation of the corresponding fluorescent protein in response. The intensity of fluorescence produced by our circuits is therefore indicative of the miRNA levels in the body fluid. To help reduce costs, we designed and built a £4 combined fluorometer and densitometer, coined a luminometer, to cheaply quantify the fluorescence from our circuit without a plate reader. This allows our test to be used in the field and in less developed countries.
In a clinical implementation, miRNAs extracted from blood serum would be added to a cell free system containing an excess of our amalgamation. The unique toehold switches would detect specific miRNAs and regulate translation of the corresponding fluorescent protein in response. The intensity of fluorescence produced by our circuits is therefore indicative of the miRNA levels in the body fluid. To help reduce costs, we designed and built a £4 combined fluorometer and densitometer, coined a luminometer, to cheaply quantify the fluorescence from our circuit without a plate reader. This allows our test to be used in the field and in less developed countries.
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