Difference between revisions of "Team:Berlin diagnostX/Design"
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| − | <h3 class="text-center igem_blue mt-4"><strong> | + | <h3 class="text-center igem_blue mt-4"><strong>Establishing the screening pipeline</strong></h3> |
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| − | <h5 class="text-left igem_blue pb-1"> | + | <h5 class="text-left igem_blue pb-1">Optimizing the Cell Free Expression System </h5> |
| − | <p class="text-justify"><em> | + | <p class="text-justify">The <em>PureExpress®-System </em> is composition of completely recombinant molecules to effectively transcribe any form of DNA and translate the produced mRNA.
To use this system for field diagnostics we figured that it has to meet certain requirements: |
| + | Heat stability up to a temperature of 50° C | ||
| + | High efficiency in low volumes and smaller concentrations | ||
| + | Stability towards change in electrolyte concentration and addition of foreign molecules | ||
| + | Storability | ||
| + | We successfully verified the outstanding function of the system under these conditions.
Especially noteworthy, we were able to create significant color change in a volume of 2,5 uL which is 10% of the volume recommended by the manufacturer. Furthermore, we could show that the system is still highly active in concentrations of 40-50%. This makes it possible to elute it with patient samples without tempering with its functionality.
Last but not least, the system is not very vulnerable to a fair amount of possibly interfering molecules like for example the chlorphenol-red we use as reporter assay. | ||
| + | We concluded that the PureExpress® - System is most suitable for our essay; also due to the fact, that it composed of recombinant parts instead of cell lysate and so has very few variability.
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Revision as of 23:08, 29 October 2017
Establishing the screening pipeline
Optimizing the Cell Free Expression System
The PureExpress®-System is composition of completely recombinant molecules to effectively transcribe any form of DNA and translate the produced mRNA. To use this system for field diagnostics we figured that it has to meet certain requirements: Heat stability up to a temperature of 50° C High efficiency in low volumes and smaller concentrations Stability towards change in electrolyte concentration and addition of foreign molecules Storability We successfully verified the outstanding function of the system under these conditions. Especially noteworthy, we were able to create significant color change in a volume of 2,5 uL which is 10% of the volume recommended by the manufacturer. Furthermore, we could show that the system is still highly active in concentrations of 40-50%. This makes it possible to elute it with patient samples without tempering with its functionality. Last but not least, the system is not very vulnerable to a fair amount of possibly interfering molecules like for example the chlorphenol-red we use as reporter assay. We concluded that the PureExpress® - System is most suitable for our essay; also due to the fact, that it composed of recombinant parts instead of cell lysate and so has very few variability.
RNA sequencing
Taenia solium, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point [1]. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis.
The bioinformatics behind our switch design
In silico design
Taenia solium, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point [1]. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis.
The switch synthesis
Extension PCR
Taenia solium, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point [1]. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis.
Nested PCR
Taenia solium, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point [1]. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis.
Colony PCR
Taenia solium, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point [1]. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis.
DNA Clean Up (via column & gel extraction)
Taenia solium, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point [1]. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis.
Gel electrophoresis
Taenia solium, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point [1]. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis.
The Idea
The Tapeworm Taenia solium
Taenia solium, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point [1]. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis.
The test will be based on a newly developed method called toehold switch sensors. This method has already been successfully applied to the diagnosis of the Zika virus [3]. We are therefore convinced that toehold switch sensors will also be useful in detecting T. solium. In order to develop our test, we first need to achieve two things: The test will be based on a newly developed method called toehold switch sensors. This method has already been successfully applied to the diagnosis of the Zika virus [3]. We are therefore convinced that toehold switch sensors will also be useful in detecting T. solium. In order to develop our test, we first need to achieve two things:
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The Experiments
Production of Toehold Switch Sensors
Taenia solium, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point [1]. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis.
Toehold Switch Sensor Test
Taenia solium, otherwise known as pork tapeworm, is a parasite with potentially serious health effects in humans. It infects millions of people worldwide, leading to severe brain diseases, blindness, epilepsy, and death. Countries where where pork production and consumption are coupled with poor hygiene are the worst affected, including large areas of Latin America, Sub-Saharan Africa, and South- and East-Asia, where over 14% of people have experienced an infection at some point [1]. However, a clear picture of how many people are currently infected by T. solium does not exist, due to the current difficulty in making a diagnosis.
Application
Überschrift
The synthetic biology experiments described here are particularly important for participation in the iGEM competition. Simultaneously, a second team will work on the recovery of T. solium-RNA from patient stool samples in order to make our test applicable for medical purposes. We are currently forming the necessary partnerships for this goal: A working group in Nairobi has already committed to providing us with relevant material. However, these experiments will be conducted in a properly equipped laboratory, independent from the previously described experiments.
