For our cloning experiments and the development of our prime editing complexes, we have amplified various plasmids in <i>E. coli</i> K-12 strains (DH5α,10-Beta). When working with microbial strains such as <i>E. coli</i> K-12 strains, it's important to consider potential risks associated with their use, even though they are generally regarded as safe in laboratory settings. All experiments were performed under strict S1 conditions, following all relevant safety protocols. Below you will find an overview of the <i>E. coli</i> K-12 strains for our cloning experiments, submitted by us as a check-In and the specific safety measures:
</p>
<p>
<strong><i>E. coli K-12</i> strains (DH5α, 10-Beta):</strong> Although these strains are non-pathogenic and have been modified to minimize the risk of spreading antibiotic resistance, there remains a low risk of horizontal gene transfer, where genetic material could be transferred to other microorganisms, potentially leading to the spread of resistance genes or other traits. If accidentally released into the environment, <i>E. coli</i> K-12 strains could potentially interact with native microbial communities. While they are typically outcompeted in natural environments, there's a remote possibility of ecological disruption, particularly in microenvironments where they could find a niche.While these strains are non-virulent, they still pose a minimal risk to humans, particularly immunocompromised individuals, through accidental ingestion or inhalation in a laboratory setting.
</p>
<p>
We submitted the yeast strain <i>Pichia pastoris</i> (SMD1163) for the protein expression of Fanzor.
</p>
<p>
<strong><i>Pichia pastoris</i> (SMD1163):</strong><i>Pichia pastoris</i> (SMD1163) is a widely used yeast strain for the expression of recombinant proteins. It is characterized by a methanol-inducible expression system (AOX1 promoter) and high cell growth rates, which makes it ideal for industrial applications. The strain can be easily genetically manipulated and can perform post-translational modifications, which supports correct protein production.
When working with <i>Pichia pastoris</i> (SMD1163), various safety-relevant aspects must be observed. Although the organism is considered non-pathogenic and biologically safe (S1), skin contact and aerosol formation should be avoided to minimize the risk of infection or allergic reactions. When using genetically modified strains, it is important to follow the relevant GMO guidelines to prevent uncontrolled release. In addition, handling chemicals such as methanol requires special precautions as they are toxic and highly flammable. The disposal of cell cultures and waste must also be carried out in accordance with biosafety regulations, especially in the case of genetically modified organisms.
</p>
<Collapsibleid="Checkcloning"open={false}title="open to see full article">
<p>
For our cloning experiments and the development of our prime editing complexes, we have amplified various plasmids in <i>E. coli</i> K-12 strains (DH5α,10-Beta). When working with microbial strains such as <i>E. coli</i> K-12 strains, it's important to consider potential risks associated with their use, even though they are generally regarded as safe in laboratory settings. All experiments were performed under strict S1 conditions, following all relevant safety protocols. Below you will find an overview of the <i>E. coli</i> K-12 strains for our cloning experiments, submitted by us as a check-In and the specific safety measures:
</p>
<p>
<strong><i>E. coli K-12</i> strains (DH5α, 10-Beta):</strong> Although these strains are non-pathogenic and have been modified to minimize the risk of spreading antibiotic resistance, there remains a low risk of horizontal gene transfer, where genetic material could be transferred to other microorganisms, potentially leading to the spread of resistance genes or other traits. If accidentally released into the environment, <i>E. coli</i> K-12 strains could potentially interact with native microbial communities. While they are typically outcompeted in natural environments, there's a remote possibility of ecological disruption, particularly in microenvironments where they could find a niche.While these strains are non-virulent, they still pose a minimal risk to humans, particularly immunocompromised individuals, through accidental ingestion or inhalation in a laboratory setting.
</p>
<p>
We submitted the yeast strain <i>Pichia pastoris</i> (SMD1163) for the protein expression of Fanzor.
</p>
<p>
<strong><i>Pichia pastoris</i> (SMD1163):</strong><i>Pichia pastoris</i> (SMD1163) is a widely used yeast strain for the expression of recombinant proteins. It is characterized by a methanol-inducible expression system (AOX1 promoter) and high cell growth rates, which makes it ideal for industrial applications. The strain can be easily genetically manipulated and can perform post-translational modifications, which supports correct protein production.
When working with <i>Pichia pastoris</i> (SMD1163), various safety-relevant aspects must be observed. Although the organism is considered non-pathogenic and biologically safe (S1), skin contact and aerosol formation should be avoided to minimize the risk of infection or allergic reactions. When using genetically modified strains, it is important to follow the relevant GMO guidelines to prevent uncontrolled release. In addition, handling chemicals such as methanol requires special precautions as they are toxic and highly flammable. The disposal of cell cultures and waste must also be carried out in accordance with biosafety regulations, especially in the case of genetically modified organisms.
</p>
</Collapsible>
<H4text="Check-in for Testing in cell lines "></H4>
<p>
In our project, we paid attention to safety at every step, especially when working with specific <ahref="methods?scrollTo=Cell Culture">cell lines</a>. All experiments were performed under strict S1 conditions, following all relevant safety protocols. Given the sensitivity of the human cell lines we used, we placed great emphasis on controlled and well-designed workflows. All transfections were performed in our own transfection laboratory to ensure a high level of safety and compliance. Below you will find an overview of the cell lines submitted by us as a checkin and the specific safety measures:
</p>
<p>
<strong>HEK293 cell line: </strong>HEK 293 (Human Embryonic Kidney 293) cells are an immortal cell line originally derived from the kidney cells of a human embryo. They are characterized by their fast division rate and high transfection efficiency, which makes them a popular model in biomedical research. For our studies, the basic HEK293 cells were provided to us by the Cellular and Molecular Biotechnology Group at Bielefeld University, headed by Prof. Dr. Kristian Müller. Prof. Dr. Müller is also one of the Principal Investigators of our team. We use this cell line in our proof-of-concept studies and for testing the Prime Editing Guide pegRNA (pegRNA) to evaluate the efficiency and functionality of our constructs.
</p>
<p>
<strong>HEK293T-3HA-CFTR cell line: </strong>The HEK293T-3HA-CFTR cell line is based on HEK293T cells expressing an additional tsA1609 allele of the SV40 large T antigen. This allele enables the replication of vectors containing the SV40 origin of replication. In addition to the native CFTR gene, which is not expressed in HEK cells, the HEK293T-3HA-CFTR cell line from Leuven carries another copy of the CFTR gene embedded in an expression cassette. This cassette contains a CMV promoter, which is derived from the human cytomegalovirus and is frequently used for the overexpression of genes in human cells. In addition, the cassette contains a puromycin resistance gene that is co-expressed with CFTR, allowing continuous selection of CFTR-expressing cells.
</p>
<p>
<strong>HEK293T-3HA-F508del-CFTR cell line:</strong> The HEK293T-3HA-F508del-CFTR cell line is a modified HEK293T cell line that carries the F508del mutation in the CFTR gene, which is responsible for the most common mutation in cystic fibrosis. This mutation leads to a defective CFTR protein that impairs the normal function of the chloride channel. The cell line is therefore ideal for studying the effects of this mutation and for evaluating potential therapies for cystic fibrosis.
</p>
<p>
<strong>CFBE41o- cell line:</strong> The CFBE41o- cell line, derived from the bronchial epithelial cells of a cystic fibrosis patient, is homozygous for the F508del-CFTR mutation and was essential for our cystic fibrosis research. A reduced CFTR expression level is present. The cell line carries the CFTR defect and can therefore represent a patient with CF. The cell line is used to test our mechanism. These cells were immortalized with a replication-defective plasmid that retains their physiological properties.
When working with the HEK293T and CFBE41o- cell lines, it’s important to consider the minimal risks associated with their use. While not harmful on their own, the genetic modifications in HEK293T cells require careful handling to prevent accidental release or exposure. These cells, engineered to overexpress CFTR, including the F508del mutation, necessitate strict safety measures like regular monitoring and proper waste disposal to comply with S1 laboratory standards. Similarly, CFBE41o- cells, due to their genetic modifications and disease relevance, require careful handling to avoid cross-contamination and ensure biosafety.
</p>
<p>
<strong>Human nasal epithelial cells (hNECs):</strong> Human nasal epithelial cells (hNECs) were harvested using a nasal brush, a minimally invasive procedure, and cultured in air-liquid interface (ALI) cultures to model the airway epithelium. Human nasal epithelial cells (hNECs) were obtained using a nasal brush, a minimally invasive technique, and then cultured in air-liquid interface (ALI) cultures to model the airway epithelium. Using these primary cultures, derived from donors with airway diseases such as cystic fibrosis, we were able to simulate the in vivo conditions of such diseases.
Due to the sensitive nature of these primary human cells, we performed all experiments with hNECs in our S2 laboratory, where increased safety precautions were taken. This included strict safety controls, safe handling of samples and proper disposal of materials after testing. In particular, the hNECs underwent HHH (Triple H: HIV, HCV and HBV) testing to ensure that no contamination occurred during sample collection or experimentation. These tests included sterility testing, viability assessments and contamination testing to ensure the safety and integrity of both the samples and the laboratory environment. After a negative HHH test, the primary cultures can be treated as S1. In addition, the nasal epithelial cells were handled with the utmost care during collection, ensuring that all procedures were performed under sterile conditions to avoid any risk of contaminationFor this purpose, the intensive examination of ethical questions was fundamental and a constant companion of our project. The numerous results from the interviews in the areas of: Ethics, storage and training in the handling of samples have been summarized in a guideline for patient consent for Germany and are intended to provide iGEM teams with the scope, critical examination and observance of iGEM rules, international and national guidelines.
</p>
<Collapsibleid="CheckcellLines"open={false}title="open to see full results">
<p>
In our project, we paid attention to safety at every step, especially when working with specific <ahref="methods?scrollTo=Cell Culture">cell lines</a>. All experiments were performed under strict S1 conditions, following all relevant safety protocols. Given the sensitivity of the human cell lines we used, we placed great emphasis on controlled and well-designed workflows. All transfections were performed in our own transfection laboratory to ensure a high level of safety and compliance. Below you will find an overview of the cell lines submitted by us as a checkin and the specific safety measures:
</p>
<p>
<strong>HEK293 cell line: </strong>HEK 293 (Human Embryonic Kidney 293) cells are an immortal cell line originally derived from the kidney cells of a human embryo. They are characterized by their fast division rate and high transfection efficiency, which makes them a popular model in biomedical research. For our studies, the basic HEK293 cells were provided to us by the Cellular and Molecular Biotechnology Group at Bielefeld University, headed by Prof. Dr. Kristian Müller. Prof. Dr. Müller is also one of the Principal Investigators of our team. We use this cell line in our proof-of-concept studies and for testing the Prime Editing Guide pegRNA (pegRNA) to evaluate the efficiency and functionality of our constructs.
</p>
<p>
<strong>HEK293T-3HA-CFTR cell line: </strong>The HEK293T-3HA-CFTR cell line is based on HEK293T cells expressing an additional tsA1609 allele of the SV40 large T antigen. This allele enables the replication of vectors containing the SV40 origin of replication. In addition to the native CFTR gene, which is not expressed in HEK cells, the HEK293T-3HA-CFTR cell line from Leuven carries another copy of the CFTR gene embedded in an expression cassette. This cassette contains a CMV promoter, which is derived from the human cytomegalovirus and is frequently used for the overexpression of genes in human cells. In addition, the cassette contains a puromycin resistance gene that is co-expressed with CFTR, allowing continuous selection of CFTR-expressing cells.
</p>
<p>
<strong>HEK293T-3HA-F508del-CFTR cell line:</strong> The HEK293T-3HA-F508del-CFTR cell line is a modified HEK293T cell line that carries the F508del mutation in the CFTR gene, which is responsible for the most common mutation in cystic fibrosis. This mutation leads to a defective CFTR protein that impairs the normal function of the chloride channel. The cell line is therefore ideal for studying the effects of this mutation and for evaluating potential therapies for cystic fibrosis.
</p>
<p>
<strong>CFBE41o- cell line:</strong> The CFBE41o- cell line, derived from the bronchial epithelial cells of a cystic fibrosis patient, is homozygous for the F508del-CFTR mutation and was essential for our cystic fibrosis research. A reduced CFTR expression level is present. The cell line carries the CFTR defect and can therefore represent a patient with CF. The cell line is used to test our mechanism. These cells were immortalized with a replication-defective plasmid that retains their physiological properties.
When working with the HEK293T and CFBE41o- cell lines, it’s important to consider the minimal risks associated with their use. While not harmful on their own, the genetic modifications in HEK293T cells require careful handling to prevent accidental release or exposure. These cells, engineered to overexpress CFTR, including the F508del mutation, necessitate strict safety measures like regular monitoring and proper waste disposal to comply with S1 laboratory standards. Similarly, CFBE41o- cells, due to their genetic modifications and disease relevance, require careful handling to avoid cross-contamination and ensure biosafety.
</p>
<p>
<strong>Human nasal epithelial cells (hNECs):</strong> Human nasal epithelial cells (hNECs) were harvested using a nasal brush, a minimally invasive procedure, and cultured in air-liquid interface (ALI) cultures to model the airway epithelium. Human nasal epithelial cells (hNECs) were obtained using a nasal brush, a minimally invasive technique, and then cultured in air-liquid interface (ALI) cultures to model the airway epithelium. Using these primary cultures, derived from donors with airway diseases such as cystic fibrosis, we were able to simulate the in vivo conditions of such diseases.
Due to the sensitive nature of these primary human cells, we performed all experiments with hNECs in our S2 laboratory, where increased safety precautions were taken. This included strict safety controls, safe handling of samples and proper disposal of materials after testing. In particular, the hNECs underwent HHH (Triple H: HIV, HCV and HBV) testing to ensure that no contamination occurred during sample collection or experimentation. These tests included sterility testing, viability assessments and contamination testing to ensure the safety and integrity of both the samples and the laboratory environment. After a negative HHH test, the primary cultures can be treated as S1. In addition, the nasal epithelial cells were handled with the utmost care during collection, ensuring that all procedures were performed under sterile conditions to avoid any risk of contaminationFor this purpose, the intensive examination of ethical questions was fundamental and a constant companion of our project. The numerous results from the interviews in the areas of: Ethics, storage and training in the handling of samples have been summarized in a guideline for patient consent for Germany and are intended to provide iGEM teams with the scope, critical examination and observance of iGEM rules, international and national guidelines.
</p>
</Collapsible>
<H4text="Check-in for Delivery "></H4>
<p>
Our finished construct is designed to be delivered into the lung via an inhaler using lipid nanoparticles (LNPs). To be more spezific a selective organ-targeting (SORT)- LNPs were developed to deliver mRNA specifically to the lung, with special measures taken to increase biocompatibility and safety. Since the LNP composition is very specific and also differs from other formulas, we submitted the LNP as a checkin:
</p>
<p>
<strong>LNP:</strong> These LNPs are then taken up by epithelial cells through endocytosis, releasing the construct into the cytosol. We carefully evaluated the potential risks, including unintended immune responses and the need for precise dosing to minimize side effects. In addition, we have conducted an in-depth analysis of the dual-use potential of our technology. Dual-use refers to the possibility that scientific advances can be used for both civilian and military purposes. Therefore, we have implemented strict safety protocols and ethical guidelines to ensure that our technology is used exclusively for peaceful and therapeutic applications.
</p>
<Collapsibleid="CheckDelivery"open={false}title="open to see full article">
<p>
Our finished construct is designed to be delivered into the lung via an inhaler using lipid nanoparticles (LNPs). To be more spezific a selective organ-targeting (SORT)- LNPs were developed to deliver mRNA specifically to the lung, with special measures taken to increase biocompatibility and safety. Since the LNP composition is very specific and also differs from other formulas, we submitted the LNP as a checkin:
</p>
<p>
<strong>LNP:</strong> These LNPs are then taken up by epithelial cells through endocytosis, releasing the construct into the cytosol. We carefully evaluated the potential risks, including unintended immune responses and the need for precise dosing to minimize side effects. In addition, we have conducted an in-depth analysis of the dual-use potential of our technology. Dual-use refers to the possibility that scientific advances can be used for both civilian and military purposes. Therefore, we have implemented strict safety protocols and ethical guidelines to ensure that our technology is used exclusively for peaceful and therapeutic applications.
