Difference between revisions of "Team:Berlin diagnostX/Experiments"
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| − | <p class="text-justify"> | + | <p class="text-justify"> Nested PCR is a method for speeding up the process of switch generation by combining the two steps of our extension-PCR in one cycling step. In order to extent the whole switch length without interruption, purification of P1-product and second PCR Set-Up. After consumption of P1-forward primer the already added P2 Primer takes over the reaction. </p> |
| + | <p class="text-justify"> Our experiments showed the suitability of nested PCR for the creation of diagnost-x toehold switches even though different to the situation described by Schuelke (1) our forward primers have equal overlaps and therefore are not suitable for modulating the annealing temperature in order to start the primers separately. Reactions with different relation in the amounts of P1 and P2 forward primer proved the relation of P1/P2=1/4 as the most reliable in sequence stability. </p> | ||
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Revision as of 00:41, 31 October 2017
Experiments
Project Management
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 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 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:
Summary: Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum.
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.
The preliminary work
RNA isolation
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.
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
Toehold switches were constructed by two PCR-based steps, in which the recognition and hairpin region was attached to a LacZ reporter element. The construction was amplified from a circular DNA Template.
Primer P1 and P2 are specifically designed for different Toehold switches.( Abb. 1) These include the Recognition-Structure, the characteristic Hairpin, Ribosome binding site and Linker. The Switches were constructed by ligating them to the lacZ reporter gene.
A specific third step was used to purify the preceding amplifications and also attach a T7 Promotor region.
Nested PCR
Nested PCR is a method for speeding up the process of switch generation by combining the two steps of our extension-PCR in one cycling step. In order to extent the whole switch length without interruption, purification of P1-product and second PCR Set-Up. After consumption of P1-forward primer the already added P2 Primer takes over the reaction.
Our experiments showed the suitability of nested PCR for the creation of diagnost-x toehold switches even though different to the situation described by Schuelke (1) our forward primers have equal overlaps and therefore are not suitable for modulating the annealing temperature in order to start the primers separately. Reactions with different relation in the amounts of P1 and P2 forward primer proved the relation of P1/P2=1/4 as the most reliable in sequence stability.
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 Toehold Switch Sensor Test
Standard screening in liquid phase
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.
CFE on membrane trials (BSA vs Whatman membrane)
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.
Taenia solium model
RNA Library
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.
Optimizing of our screening pipeline
Primer efficiency analysis & PCR characterization (P2 vs P3)
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.
Optimizing CFE
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.
Other Experiments
TA Cloning
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.
Fusion Cloning
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.
Mutagenesis
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.
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:
Summary: Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum.
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.
Protocols
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 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 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:
Summary: Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum.
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.
