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+++ b/wiki/menu.html
@@ -39,7 +39,7 @@
<ul class="dropdown-menu bg-dark" aria-labelledby="navbarDropdown">
<li><a class="dropdown-item" href="{{ url_for('pages', page='contribution') }}">Contribution</a></li>
<li><a class="dropdown-item" href="{{ url_for('pages', page='description') }}">Description</a></li>
- <li><a class="dropdown-item" href="{{ url_for('pages', page='design') }}">Design</a></li>
+ <!-- <li><a class="dropdown-item" href="{{ url_for('pages', page='design') }}">Design</a></li> -->
<li><a class="dropdown-item" href="{{ url_for('pages', page='engineering') }}">Engineering</a></li>
<li><a class="dropdown-item" href="{{ url_for('pages', page='modeling') }}">Modeling</a></li>
<!-- <li><a class="dropdown-item" href="{{ url_for('pages', page='experiments') }}">Experiments</a></li> -->
diff --git a/wiki/pages/description.html b/wiki/pages/description.html
index fca19a5b84de5cf11900dd874455db5c4c5ceee8..169437b8da705aab3b3e6361a261340295f703a1 100644
--- a/wiki/pages/description.html
+++ b/wiki/pages/description.html
@@ -13,6 +13,10 @@
<h4>Project Description USP-Brazil 2024</h4>
<div class="bd-callout bd-callout-info">
<ul>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem15.webp">
+ <figcaption>Figure 1. Glycan profiles in mammalian, plant, insect, archaeal, Saccharomyces cerevisiae and Escherichia coli proteins</figcaption>
+ </figure>
Eukaryotes have evolved the ability to modify their proteins by linking polysaccharide groups to asparagine residues, with these sugars being essential for the proper folding and activity of glycoproteins. In humans, more than 50% of proteins are glycosylated, rising up to 70% in certain tissues. The complex pathways required for the assembly and transfer of glycans to proteins pose significant challenges when adapting these processes to prokaryotic models, such as Escherichia coli, the most commonly used organism for heterologous protein production. In contrast, Saccharomyces cerevisiae possesses a functional machinery for glycoprotein production. However, it predominantly produces hyper-mannose-type glycosylation, which is immunogenic in humans, making it unsuitable for the production of therapeutic glycoproteins.
</ul>
</div>
@@ -25,8 +29,14 @@
<ul>
<p><strong>Why Glycoproteins?</strong></p>
In addition to humans, human viruses can benefit from the glycosylation machinery of host cells to modify their proteins, a critical feature for completing their infection cycles. This glycosylation aids in evading the host immune response and enhancing viral entry and replication. During the 2020 pandemic, Prof. Dr. Cristiane Rodrigues Guzzo adapted the projects in her research group to collaborate with the Brazilian ReveVirus MCTI program. Together, they expressed and purified proteins from SARS-CoV-2 using Escherichia coli, distributing these proteins to other research groups. This initiative created a collaborative network of researchers focused on understanding the physiology of these proteins and their potential applications in developing diagnostic tests.
+ <img src="https://static.igem.wiki/teams/5428/imagem16.webp">
<p>However, not all viral proteins can be adequately expressed in E. coli due to its lack of glycosylation machinery, which affects protein folding. For instance, the Spike protein that mediates membrane fusion and virus entry in cells is insoluble in E. coli, making it impossible to produce by this system. This way, the Spike protein could not be distributed for other groups to study. </p>
<p>Additionally, during a discussion with Prof. Dr. LuÃs Carlos de Souza Ferreira, who focuses on developing vaccines against human viruses, we discovered that this issue is longstanding. The absence of a glycosylation pathway in E. coli limits the production of these proteins to eukaryotic cells, leading to increased costs and requiring long incubation periods to produce small amounts of proteins.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem17.webp">
+ <figcaption>Figure 2. Glycoprotein folding scheme.
+ </figcaption>
+ </figure>
</ul>
</div>
</div>
@@ -38,9 +48,24 @@
<ul>
<p><strong>Motivation: Engineering E. coli and S. cerevisiae to produce human glycoproteins</strong></p>
<p>Glycosylation in eukaryotes begins with the assembly of the glycan on the cytoplasmic surface of the endoplasmic reticulum (ER). After forming the Man5GlcNAc2 structure, the glycan is flipped into the lumen of the ER, where it undergoes further modification by acquiring additional sugars. This modified glycan is then transferred to the target protein by oligosaccharyltransferases. Once glycosylated, the protein is delivered to the Golgi apparatus via vesicles, where it receives the final sugar additions before being secreted.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem18.webp">
+ <figcaption>Figure 3. Glycosylation pathway in eukaryotes. Glycan assembly and transference in the endoplasmic reticulum, delivery, and final modifications in the goldi apparatus. Source: <a href = "https://doi.org/10.3389/fpls.2024.1349064">https://doi.org/10.3389/fpls.2024.1349064</a>
+ </figcaption>
+ </figure>
<p>In contrast, prokaryotic cells lack organelles like the endoplasmic reticulum and Golgi apparatus. However, some species have evolved glycosylation machinery that assembles glycans on the cytoplasmic face of the inner membrane. Once synthesized, these glycans are recognized and flipped to the periplasm, where they can be transferred en bloc to the target protein by an oligosaccharyltransferase. This feature is shared among both bacteria and archaea.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem19.webp">
+ <figcaption>Figure 4. Glycosylation pathway in Campylobacter jejuni. Source: <a href = "https://doi.org/10.1021/ac9013622">https://doi.org/10.1021/ac9013622</a>
+ </figcaption>
+ </figure>
<p>Compared to cell tissue, insect, and plant expression models, which take around three weeks to complete the production and purification cycle of small protein quantities at elevated costs due to complex media requirements, E. coli and S. cerevisiae are fast-growing microorganisms widely used in biotechnological processes. These organisms can be used to produce and purify large amounts of proteins in just three to five days. Additionally, they can be cultivated in low-cost media and are not susceptible to human viruses that can contaminate cultures. Adapting both organisms for the adequate production of glycoproteins has the potential to significantly enhance the global production of these valuable products.</p>
<p>To achieve this, we must decide which glycoform we aim to produce. Initially, we wanted to adapt these organisms for assembling human-type polysaccharides with hybrid or complex glycans. However, we realized that the pathways involved in preparing precursors, assembling sugars, and transferring them to proteins are extremely complex. Then, what kind of glycan is simple to work within two years and would interest companies and academic research groups? The answer lies in the core structure, Man3GlcNac2!</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem20.webp">
+ <figcaption>Figure 5. Structure of Man3GlcNac2 and conservation among different organisms.
+ </figcaption>
+ </figure>
<p>Man3GlcNAc2 is a simple polysaccharide produced in the early stages of glycan assembly on the outer surface of the endoplasmic reticulum. Furthermore, only four proteins are required to synthesize this glycan, making it feasible to reconstruct the system in E. coli. Additionally, yeasts possess the necessary machinery to produce this structure, which can be achieved through genome knockout of genes involved in glycan extension by adding new mannose residues. </p>
<p>Another important characteristic in selecting these sugars is that they represent the conserved core shared among all the aforementioned organisms. This foundational aspect allows for future research groups to reengineer our Saccharomyces cerevisiae and Escherichia coli systems to produce the desired type of glycosylation. By adding new glycosyltransferases to the pathway — such as those found in the Golgi apparatus, which are capable of inserting additional sugars into the already glycosylated proteins — researchers can assemble the glycosylation profiles to meet specific therapeutic or research needs. This flexibility paves the way for further advancements in glycoprotein production and engineering.</p>
<p>To engineer our organisms, two completely different strategies were developed; ‘. Adaptation of the Existing Glycosylation Pathway in S. cerevisiae: This approach aims to ensure that the yeast accumulates our glycan of interest while avoiding the common challenge of engineering yeasts for glycosylation - the unintended addition of excess mannose residues due to the nonspecific activity of various mannosyltransferases involved in extending to hyper-mannose types.; and 2. Creation of a De Novo Glycosylation Pathway in E. coli: Since E. coli lacks a natural glycosylation machinery, this strategy involves building the pathway from zero, enabling the bacterium to produce our desired glycan.</p>
@@ -48,8 +73,30 @@
<p>For the bacterial part, we based our experiments on the work of DeLisa <a href="https://doi.org/10.1038%2Fnchembio.921">https://doi.org/10.1038%2Fnchembio.921</a>. To construct a glycosylation pathway in E. coli, we cloned the Alg13, Alg14, Alg1, and Alg2 genes from S. cerevisiae into a plasmid to enable glycan assembly on the cytoplasmic surface of the inner membrane. To increase the precursor availability for glycan assembly, we also cloned the ManB and ManC genes under the control of a constitutive expression promoter and knocked out the GMD gene to prevent the degradation of mannose, which could serve as a carbon source.</p>
<p>Since E. coli possesses the Wzx flippase that translocates lipopolysaccharide (LPS) precursors to the periplasmic surface, we chose to retain this component in our system to facilitate the transport of our glycans. Additionally, the WaaL protein, known for linking assembled sugars to the bacterial LPS, poses a potential concern as it may reduce the amount of glycans available in the periplasm. However, it can also serve as a tool to evaluate whether our glycan was properly produced. For our target Man3GlcNAc2, the exposed mannose residues can be stained with Alexa Fluor-488, and the presence of fluorescent cells would confirm the successful production of Man3GlcNAc2. Once confirmed, we would need to knock out WaaL to prevent the delivery of glycans to the LPS, thereby increasing the available glycans for our glycoprotein.</p>
<p>A crucial aspect of the bacterial system involves sugar transfer. Campylobacter jejuni possesses the most studied bacterial glycosylation system, featuring the PglB oligosaccharyltransferase (OST), which links glycans to proteins. PglB is extensively researched in efforts to engineer bacterial organisms capable of glycosylation. However, it differs from its eukaryotic homologs; while Stt3 OSTs recognize the N-X-S/T (where X can be any amino acid except proline) sequon, PglB specifically glycosylates asparagine residues within the D/E-X-N-X-S/T (X = any amino acid except proline) sequon. Therefore, it is necessary to adapt the sequence of our protein of interest for expression in systems utilizing PglB. This poses a challenge, particularly when working with large proteins such as the Spike protein from SARS-CoV-2, which contains 22 N-glycosylation sites. Adapting these sites could result in a significantly more negatively charged protein compared to the wild type, potentially compromising its folding and function.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem21.webp">
+ <figcaption>Figure 6. glycosylation sequons in eukaryotes and bacteria
+ </figcaption>
+ </figure>
<p>The obvious question that arose was: if we are utilizing the glycosylation machinery from yeast, why not also use the oligosaccharyltransferase (OST)? The issue lies in the organization of this complex. While bacteria like Campylobacter jejuni have a single OST responsible for transferring sugars to asparagine residues, eukaryotes rely on a complex of eight proteins that collaborate in sugar recognition and transfer. The likelihood that this complex would assemble correctly in E. coli is low, making it an unattractive option for cloning purposes.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem22.webp">
+ <figcaption>Figure 7. Membrane proteins responsible for sugar transference. PDB structures of C. jejuni (A) and Homo sapens (B) OSTs.
+ </figcaption>
+ </figure>
<p>If the eukaryotic Stt3 is not a viable option, where could we find an OST that could recognize the human glycosilation sequon, but have only one protein that makes the glycan transference? Exploring the literature, we found another group that can also perform glycosylation: archaea. These microorganisms are intriguing due to their unique cellular processes, which have adapted to extreme conditions, and glycosylation plays a crucial role in helping them resist various stressors. Surprisingly, we found that archaea can recognize the human N-X-S/T sequon and, much like bacteria, they rely on just one OST to carry out the glycosyltransferase activity: AglB. This presents a promising alternative for our research, as AglB could potentially provide a simpler and more efficient mechanism for glycosylation in our engineered systems.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem23.webp">
+ <figcaption>Figure 8. Structure and sequence recognition by archaeal OST. A. Alphafold structure of AglB from Methanothermus ferividus. B. Glycosylation sequons recognized by eukaryotes, bacteria and archaeas
+ </figcaption>
+ </figure>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem24.webp">
+ <figcaption>Figure 9. Archaeal glycan structures. Source: <a href = "https://doi.org/10.1038/nrmicro2957
+ ">https://doi.org/10.1038/nrmicro2957</a>
+
+ </figcaption>
+ </figure>
<p>Despite their promise, archaea produce glycans that are entirely different from those found in bacteria and eukaryotes. This divergence could pose a challenge, as the recognition of linking sugars is a critical factor that may hinder their transfer to the target protein. Therefore, rather than using PglB, we opted to focus on three particularly interesting oligosaccharyltransferases (OSTs) from Methanococcus voltae, Methanothermus fervidus, and Sulfolobus acidocaldarius. These OSTs were selected based on the specific substrates they recognize for transfer to proteins, allowing us to explore options that may be more compatible with our goals for glycosylation.</p>
<p>Additionally, we found that lower-order eukaryotes also possess oligosaccharyltransferases (OSTs) that can recognize human glycosylation sequons. Although these OSTs consist of a single protein, they often appear as paralogs within the chromosome. We wanted to incorporate these eukaryotic OSTs into our research; however, we reached the maximum base pairs allowed for synthesis from IDT and Twist while working on the archaeal AglBs and the proteins required for the initial stages of glycan assembly. For this reason, we decided to reserve the eukaryotic Stt3 for the second year of the project, allowing us to integrate it into our future experiments.</p>
<p>To confirm the functionality of our glycosylation system, we need to produce a glycoprotein. However, since human glycoproteins often rely on specific glycosylation profiles for activity that can be dependent of hybrid or complex type glycosilation, producing a simpler glycan could pose a risk of rendering our protein inactive. In light of this, we sought a protein that would retain its activity even with a less complex glycan. This idea was suggested by our second scientific PI, Prof. Dr. Mario Henrique de Barros: the human β-glucocerebrosidase (GCase).</p>
@@ -63,6 +110,11 @@
<li><strong>Benchmarking:</strong> The three commercially available GCase enzymes provide a reference point, enabling us to compare the enzymatic activity of our glycoprotein against currently used treatments.</li>
</ul>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem25.png">
+ <figcaption>Figure 10. Structure and glycosylation of the human GCase. A. Structure of the human GCase highlighting the glycosylated asparagine residues. B. expected profile of glycosilation in the GCase produced in our system.
+ </figcaption>
+ </figure>
<p>Selecting human GCase is of significant importance for public health in Brazil. Although Gaucher's disease is considered rare, with approximately 500 patients affected in the country, the production costs for the enzyme can be extremely high, reaching up to $300,000 per patient annually.</p>
<p>(<a href="https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2024.1433970/full#B25">https://www.frontiersin.org/journals/pharmacology/articles/10.3389/</a>)</p>
<p>GCase is provided free of charge to patients through the Unified Health System (Sistema Único de Saúde - SUS). By developing an alternative production method for this therapeutic glycoprotein using Escherichia coli, we could significantly reduce production costs, thereby alleviating the financial burden on the Brazilian healthcare system.</p>
diff --git a/wiki/pages/engineering.html b/wiki/pages/engineering.html
index bcbee5d0b365dc0e7bb794906fd07b6706aee5e8..04c2ebdda4e09e9d90644315ca032a750fcc8c1c 100644
--- a/wiki/pages/engineering.html
+++ b/wiki/pages/engineering.html
@@ -38,8 +38,10 @@
</ol>
The project's success relies on the precise integration of these components. For example, for glycosylation of human GCase, it must be directed to the appropriate cellular compartment, achievable by fusing signal peptides to guide it correctly. This integrated approach ensures that your engineered E. coli and S. cerevisiae models can efficiently produce human-compatible glycoproteins.
<p><strong>Saccharomyces cerevisiae engineering</strong></p>
+ <img src = "https://static.igem.wiki/teams/5428/imagem1.webp" style = "width: 80%; height: 80%;">
<p>The budding yeast Saccharomyces cerevisiae is an excellent choice for this iGEM project, since it’s a well-known eukaryotic model with lots of advantages for synthetic biology – and it has an endogenous glycosylation pathway! </p>
<p>Like in other eukaryotes, the glycosylation pathway in S. cerevisiae has similar initial steps that make the common core carbohydrate </p>
+ <img src = "https://static.igem.wiki/teams/5428/imagem2.webp" style = "width: 80%; height: 80%;">
<p>However, while the next steps in the human glycosylation pathway make subtle modifications, in yeast there is the generation of a hypermannosylated glycoprotein – which is immunogenic - Hence, we must modify this glycosylation pathway.</p>
<img src = "https://static.igem.wiki/teams/5428/yeast1.png" style = "width: 80%; height: 80%;">
<p></p>
@@ -50,14 +52,22 @@
<li><strong>Alg11 Deletion:</strong> This gene, involved in adding mannose residues to the cytoplasmic side of the ER, is deleted last. Since the flippase transfers the Man5GlcNAc2 to the ER lumen, removing Alg11 might interfere with this flipping process. If this proves problematic, the double mutant ΔOCH1ΔAlg3 will be used instead.</li>
</ul>
<p>This stepwise approach minimizes potential metabolic burden and growth impairment while guiding <em>S. cerevisiae</em> toward producing the simpler Man3GlcNAc2 glycan.</p>
-
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem4.webp">
+ <figcaption>Figure 1. Strategy for engineering Saccharomyces cerevisiae for the production of the polysaccharide of interest.
+ </figcaption>
+ </figure>
<p>At this stage, we anticipate two main challenges:</p>
<ol>
<li><strong>Potential Inhibition of Flippase Activity by Alg11 Deletion:</strong> The deletion of Alg11 might interfere with the flippase's ability to recognize its substrate, Man5GlcNAc2. This could lead to inefficient translocation of the glycan to the lumen of the endoplasmic reticulum, disrupting the glycosylation process.</li>
<li><strong>Nonspecific Glycosylation by Other Transferases:</strong> Even with the deletion of the target genes, there remains a risk that other glycosyltransferases in <em>S. cerevisiae</em> might nonspecifically add unwanted mannose residues to our glycan structure.</li>
</ol>
<p>To address these issues, we plan to introduce the α-1,2 mannosidase from <em>Trichoderma reesei</em> in our system. This enzyme will help trimming any excess mannose residues from Man5GlcNAc2 down to Man3GlcNAc2. By fusing an HDEL tag in the C-terminal region of this protein, we will ensure that this mannosidase remains in the endoplasmic reticulum lumen, where it can efficiently perform its function and maintain the desired glycan structure. IDT was essential for the creations of the synthetic gene coding for our codon-optimized α-1,2-mannosidase.</p>
-
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem5.webp">
+ <figcaption>Figure 2. α-1,2-mannosidase activity over Man5GlcNac2, trimming it down to Man3GlcNac2
+ </figcaption>
+ </figure>
<p>Once engineered, our yeast will be challenged to produce the selected protein, the human glucocerebrosidase (GCase). We have developed two cloning strategies for GCase expression using the shuttle vector YIp352, designed for overexpression under the control of the constitutive TEF and GPD promoters. To ensure proper targeting to the endoplasmic reticulum, an ER signal peptide tag was added to the N-terminal of GCase (yellow region), directing its delivery. Once the system is fully assembled, we will express GCase and evaluate its glycosylation efficiency, enzymatic activity, and other relevant criteria to confirm successful glycoprotein production in our engineered yeast model. Additionally, a step-tag was added to the C-terminal of GCase to facilitate its purification.</p>
<img src = "https://static.igem.wiki/teams/5428/yeast10.png" style = "width: 80%; height: 80%;">
@@ -65,17 +75,48 @@
<p><strong>Escherichia coli engineering</strong></p>
<p>Escherichia coli is currently the most widely used model organism for heterologous protein expression. Its simple prokaryotic cellular architecture, along with well-characterized metabolic pathways, has established E. coli as a key chassis for cloning and various biotechnological applications. However, when it comes to glycoproteins, E. coli presents several limitations.</p>
+ <img src = "https://static.igem.wiki/teams/5428/imagem7.webp" style = "width: 80%; height: 80%;">
<p>In contrast to eukaryotes, where post-translational modifications are common — such as in humans, where an estimated 50% to 70% of proteins are glycosylated — N-glycosylation in bacteria is a rare occurrence. Specifically, E. coli lacks an intrinsic glycosylation system, which poses a significant challenge for the heterologous expression of human proteins. As a result, this often leads to the production of insoluble, misfolded proteins that aggregate into inclusion bodies.</p>
+ <img src = "https://static.igem.wiki/teams/5428/imagem8.webp" style = "width: 80%; height: 80%;">
<p>To give E. coli the ability to synthesize our glycan, we focused on reconstructing the glycosylation assembly pathway on the cytoplasmic face of the inner membrane, given that some glcsyltransferase require their transmembrane domains for proper activity. We leveraged the native E. coli WecA protein, which can transfer GlcNAc from the precursor UDP-GlcNAc to the lipid-linker undecaprenol pyrophosphate embedded in the membrane. For the next step, the addition of the second GlcNAc residue is performed by the eukaryotic proteins Alg13 and Alg14.</p>
<p>The assembly continues with Alg1 adding the first mannose, followed by Alg2, which adds the remaining two terminal mannose residues to form Man3GlcNAc2. Once the glycan structure is complete, it will be flipped from the cytoplasmic side to the periplasmic space by Wzx, a native E. coli flippase. Wzx normally flips the precursor sugars used in LPS synthesis, but in this engineered system, it will be useful to invert the direction of the Man3GlcNAc2 glycan, making it available for glycoprotein production in the periplasmic space.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem9.webp">
+ <figcaption>Figure 3. Strategy for engineering Escherichia coli for the production of the polysaccharide of interest in the inner membrane.
+ </figcaption>
+ </figure>
<p>To enhance substrate availability for our glycosyltransferases, we will upregulate the expression of the ManB and ManC enzymes. These enzymes are crucial for the biosynthesis of UDP-mannose, which serves as a key precursor for glycan assembly. Additionally, we will knock out the GMD gene (GDP-mannose dehydratase), which is involved in mannose catabolism, to prevent the degradation of mannose and ensure an increased pool of the precursor available for glycosylation processes. </p>
<p>Initially, we aimed to integrate the glycosyltransferase genes into an operon and insert it into the E. coli chromosome at the IS5 intergenic region using the lambda red recombineering technique. However, considering the limited time for wet lab experiments this year, we opted for a more practical approach by cloning the entire operon into the pRSFDuet-1 plasmid. We placed the operon under the control of the low expression constitutive J23109 promoter (iGEM Part:BBa_J23109).</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem10.webp">
+ <figcaption>Figure 4. Empty pRSFDuet-1 map. Image created using the SnapeGene software.
+ </figcaption>
+ </figure>
<p>Despite choosing plasmid expression for now, we preserved the homology regions in our construct to facilitate future chromosomal integration in the second year of the project. To use this construct, we designed primers that would amplify the operon, excluding the homology regions, allowing us to insert it into the pRSFDuet-1 vector via Gibson assembly. Additionally, we plan to perform PCR on the pRSFDuet-1 plasmid to remove the original T7 promoter, ensuring that only the J23109 promoter regulates the expression of our glycosylation operon.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem11.webp">
+ <figcaption>Figure 5. Final construct containing the genes for precursor synthesis and glycan assembled. All of these genes were codon-optimized for E. coli BL21 DE3 before being sinthesized by IDT. Image created using the SnapGene software.
+ </figcaption>
+ </figure>
<p>The final construct would consist of one transcriptional unity containing the four glycosyltransferase genes, Alg13, Alg14, Alg1 and Alg2, followed by the ManB and ManC genes.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem12.webp">
+ <figcaption>Figure 6. Final cloned plasmid containing the whole operon for precursor synthesis and glycan assembly. Image created using the SnapGene software.
+ </figcaption>
+ </figure>
<p>Before cloning the GCase into the plasmid, we needed to ensure that our enzyme could undergo glycosylation properly. Since our system directs the final glycan to the periplasm, the enzyme must be targeted to this cellular space. While expressing our protein in the periplasm might reduce overall yield, this strategy is advantageous for complex human protein, which contains eight cysteine residues (four of them forming two disulfide bonds). Expressing it in the periplasm avoids the reducing environment of the bacterial cytoplasm, which can compromise the formation of disulfide bonds. This strategy is beneficial for other human proteins with similar disulfide bond requirements.</p>
<p>To direct the enzyme to the periplasm, we fused a PelB signal peptide to its N-terminal region. The PelB tag was chosen because it is recognized by the Sec system, which transports <strong>unfolded</strong> proteins to the periplasm. This choice is crucial, as the oligosaccharyltransferases (OSTs) in our system only recognize unfolded proteins, making the Tat system (which transports folded proteins) unsuitable. Additionally, we added a Strep-tag to the C-terminal region of the protein to facilitate its purification.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem13.webp">
+ <figcaption>Figure 7. Glycosylation pathway in the periplasm of the engineered E. coli.
+ </figcaption>
+ </figure>
<p>At this point, it would be necessary to prepare the second plasmid containing the GCase enzyme and the olygosaccharyltransferases. pETDuet-1 was chosen due to its replication origin compatible with pRSFDuet-1, and also by having two cloning sites. GCase was cloned into the first multiple cloning site (MCS) using gibson assembly. Once prepared, the second MCS must be used to integrate the olygosaccharyltransferases chosen for this work. by creating different combinations, we could finally evaluate what of these enzymas are better for this purpose.</p>
- <p>Strategy for creating different combinations of the human β-Glucocerebrosidase (GCase) with various oligosaccharyltransferases (OSTs). OST1, OST2, and OST3 correspond to the OSTs from the three selected archaea. Additionally, OST4 and OST5 (represented as OST5a and OST5b) are from the low-order eukaryotes Leishmania major and Trypanosoma brucei, respectively. Although the low-order eukaryotic OSTs will not be utilized in this year’s project (we reached the maximum synthesis provided by IDT and Twist) the constructions are prepared and will be tested in the project's second year.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem14.webp">
+ <figcaption>Figure 8. Strategy for creating different combinations of the human β-Glucocerebrosidase (GCase) with various oligosaccharyltransferases (OSTs). OST1, OST2, and OST3 correspond to the OSTs from the three selected archaea. Additionally, OST4 and OST5 (represented as OST5a and OST5b) are from the low-order eukaryotes Leishmania major and Trypanosoma brucei, respectively. Although the low-order eukaryotic OSTs will not be utilized in this year’s project (we reached the maximum synthesis provided by IDT and Twist) the constructions are prepared and will be tested in the project's second year.
+ </figcaption>
+ </figure>
<p>Finally, toconcluce our work on GCase production, we will initially focus on evaluate the protein expression. Our proof of concept for this year centers on engineering our system to yield soluble GCase, marking a significant achievement, given that past attempts to express GCase in E. coli resulted in insoluble protein formation (https://doi.org/10.1007/s12033-010-9303-4). Moving forward into the second year, we aim to assess our GCase against commercially available enzymes. The evaluation will include its efficiency in macrophage phagocytosis, lysosomal delivery, and enzymatic activity, providing a comprehensive comparison to existing treatments.</p>
</ul>
</div>
diff --git a/wiki/pages/modeling.html b/wiki/pages/modeling.html
index b5b2f6a0a44cda6d34b808b7d0bc76b2451cc72a..3ed0f5f1f9e885bf0118cafd9a350dc68549040f 100644
--- a/wiki/pages/modeling.html
+++ b/wiki/pages/modeling.html
@@ -8,9 +8,52 @@
<div class="row mt-4 justify-content-center">
<div class="col-lg-8 mx-auto">
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+ <h4>How can our system impact the life cycle of E. coli?</h4>
+ <div class="bd-callout bd-callout-info">
+<p>Since the glycosylation process will occur in the periplasm of our engineered E. coli strain, it is essential to consider the insertion of glycans into nonspecific protein sites that are also transported by the SEC system. As Oligosaccharyltransferases (OST) act on non-folded proteins, we do not need to worry about proteins transported to the periplasm by the Tat system, which only transports already folded proteins.</p>
+<p>To identify a list of proteins that can be glycosylated by our system, which will be introduced into the E. coli BL21(DE3) strain, we compiled a list of all the coding sequences (CDS) from this strain’s genome (NCBI Reference Sequence: NZ_CP053602.1). We then conducted a search for proteins containing one or more motifs of the type “N[^P][ST]â€. This search yielded a FASTA file containing 2,565 proteins, each with at least one glycosylation site.</p>
+<p>Since our goal is to identify only the proteins that can be transported to the periplasm by the SEC system, we used the SignalP 5.0 software (1) to search for signal peptides recognized by this system. We used as input the glycosylation motif-containing proteins from E. coli BL21(DE3).</p>
+<p>The table resulting from the signal peptide search was converted into a Pandas DataFrame (2), from which we filtered the proteins predicted as SP (Sec/SPI) or LIPO (Sec/SPII), the two types of signal peptides recognized and imported by the SEC system. After filtering, we obtained a list of 410 proteins that possess glycosylation sites and are imported into the periplasm by the SEC system.</p>
+<p>With this data, we intend to determine which of these proteins may have their functions altered due to the potential inserted glycosylations.</p>
+<p>REFERENCES</p>
+<ol>
+ <li>José Juan Almagro Armenteros, Konstantinos D. Tsirigos, Casper Kaae Sønderby, Thomas Nordahl Petersen, Ole Winther, Søren Brunak, Gunnar von Heijne and Henrik Nielsen. Nature Biotechnology, 37, 420-423, doi:10.1038/s41587-019-0036-z (2019)</li>
+ <li>The pandas development team. (2024). pandas-dev/pandas: Pandas (v2.2.3). Zenodo. <a href="https://doi.org/10.5281/zenodo.13819579">https://doi.org/10.5281/zenodo.13819579</a></li>
+</ol>
+ </div>
+ </div>
+ </div>
+</div>
+
+<div class="row mt-4 justify-content-center">
+ <div class="col-lg-8 mx-auto">
+ <h4>Introduction to protein molecular dynamics?</h4>
+ <div class="bd-callout bd-callout-info">
+ <p>Molecular dynamics (MD) simulations are used to predict how each atom of a macromolecule will move over time. These predictions are based on physical laws that, combined, govern the movement of the system. In this regard, force fields are considered, which are generally divided into bonded (or intramolecular) and non-bonded (or intermolecular) interactions [1, 2]. Each of these interactions is modeled with an appropriate function, which is adjusted based on experimental data or quantum calculations. Bonded interactions describe the dynamics between atoms that are directly connected by chemical bonds and are described as bond stretching energy (modeled as a harmonic function), bond angle energy (modeled as angular deformation between three atoms connected by two bonds), and torsional or dihedral energy (which describes rotation around a chemical bond). Conversely, non-bonded interactions describe forces between atoms or molecules that are not directly chemically bonded and are responsible for phenomena such as van der Waals interactions and electrostatic interactions. Van der Waals interactions are described by Lennard-Jones energy, while electrostatic interactions are determined by electrostatic energy (calculated from Coulomb's law).</p>
+ <p>At the molecular level, molecular dynamics simulations provide detailed insights into important biomolecular processes, such as conformational changes, ligand interactions, and the study of protein folding. Furthermore, molecular dynamics has the ability to predict how biomolecules will respond, at the atomic level, to perturbations such as mutations, post-translational modifications (such as phosphorylations and glycosylation), protonations, or the addition/removal of a ligand. These simulations are often combined with a wide range of experimental structural biology techniques such as X-ray crystallography, cryo-electron microscopy (cryo-EM), nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and Förster resonance energy transfer (FRET).</p>
+ <p>In the context of molecular dynamics (MD) applied to the study of biophysical and structural processes, we investigated the wild-type β-glucocerebrosidase (GCaseWT) and two mutants, N19E (GCaseMut1) and N19D (GCaseMut2). Our study focused on the stability of glycosylation in GCaseWT and its mutants, maintaining Man3GlcNac2 glycosylations at the N59, N146, and N270 residues, as seen in the workflow (Figure 1).</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem26.webp">
+ <figcaption>Figure 1. Workflow of this study. Homology modeling and molecular dynamics simulations of the GCaseWT and its variants N19E and N19D.
+ </figcaption>
+ </figure>
+ <p>To construct accurate structural models, the imiglucerase structure obtained from X-ray crystallography (PDB ID 2J25) was used as a template in the Swiss-Model web server. Mutant structures with the N19E and N19D substitutions were generated by introducing these mutations into the Swiss-Model sequence. Glycosylation was performed using the Charmm-Gui web server (https://www.charmm-gui.org/). The system was equilibrated using NVT and NPT ensembles, followed by a 100 ns molecular dynamics simulation. The molecular dynamics results are shown in Figures 1 and 2.</p>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem27.webp">
+ <figcaption>Figure 2. Radius of gyration (Rg) and RMSD (root-mean-square deviation) analysis for GCase variants. The plot shows the radius of gyration (Rg) and RMSD for GCase Wild Type (WT), Mutant 1 (Mut1), and Mutant 2 (Mut2) over the simulation time. The Rg is used to evaluate the compactness and overall structural stability of the proteins, where consistent values indicate stable conformations. The RMSD provides a measure of structural deviation from the initial configuration, with smaller fluctuations indicating higher stability. Together, these metrics offer information into the conformational stability of the GCase variants during the simulation.
+ </figcaption>
+ </figure>
+ <figure>
+ <img src="https://static.igem.wiki/teams/5428/imagem28.webp">
+ <figcaption>Figure 3. Root-mean-square fluctuation (RMSF) analysis. The RMSF values for GCase Wild Type (WT), Mutant 1 (Mut1), and Mutant 2 (Mut2) are presented, illustrating the backbone fluctuations within each variant over the simulation period. RMSF serves as a metric to assess the degree of movement of individual atoms, indicating regions of stability and flexibility within the protein structure. Higher RMSF values suggest greater structural flexibility, while lower values reflect less structural flexibility.
+ </figcaption>
+</figure>
+<p>Overall, our results indicate that the mutants exhibited a stable radius of gyration throughout the simulation, providing strong evidence that the predicted and simulated structures closely resemble their potential real structures. The RMSD values of the three proteins were comparable, allowing us to conclude that the apo-state of GCase and its mutants shows good conformational stability. Minor variations observed are due to statistical fluctuations, which did not exceed 3 Ã…, further supporting the proteins' stable conformational behavior.</p>
+<p>In addition to demonstrating good stability, it also was observed that the average fluctuations of the residues were preserved regardless of the mutations. This suggests that the protein may retain its functionality even in the presence of these alterations. The movies show the conformational changes of GCaseWT and its mutants N19E and N19D. However, it is essential to conduct further experiments to validate these results. Performing experimental studies will confirm whether the enzyme and its mutants exhibit efficient binding affinity, supporting the hypothesis that the protein's functionality is not compromised by these mutations. This investigation is key for understanding the functional implications of the mutations and their potential impact on biotechnological and therapeutic applications.</p>
+<p>REFERENCES</p>
+<p>[1] Schaffer LV, Ideker T. Mapping the multiscale structure of biological systems. Cell Syst. 2021 Jun 16;12(6):622-635. doi: 10.1016/j.cels.2021.05.012. PMID: 34139169; PMCID: PMC8245186.</p>
+<p>[2] Sinha S, Tam B, Wang SM. Applications of Molecular Dynamics Simulation in Protein Study. Membranes (Basel). 2022 Aug 29;12(9):844. doi: 10.3390/membranes12090844. PMID: 36135863; PMCID: PMC9505860.</p>
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diff --git a/wiki/pages/safety.html b/wiki/pages/safety.html
index 29b28a79d917427e70eaaa3c2cf169e66b03aa5c..f23781f756dca45d1a3157f63ec5449c47517854 100644
--- a/wiki/pages/safety.html
+++ b/wiki/pages/safety.html
@@ -1,7 +1,7 @@
{% extends "layout.html" %}
{% block title %}Safety{% endblock %}
-{% block lead %}Describe all the safety issues of your project.{% endblock %}
+{% block lead %}{% endblock %}
{% block page_content %}