RNA Isolation

Toehold switch sensors are based on synthetic biology. Essentially, they are RNA molecules which code for a reporter protein. They consist of a specific toehold sequence, a ribosome-binding site (which is important for the production of proteins) and a sequence for the reporter protein. The reporter protein can only be produced if the sensor has bonded with its specific target RNA sequence [4]. When producing the sensors, we can select both the toehold sequence and the reporter protein with complete flexibility to match our needs. In this way we can fashion our Wormspotter so that it only sends a desired signal when it binds to RNA molecules specific to T. solium. We are planning to use T. solium-specific RNA sequences for the toehold sequence and beta-Galactosidase as a reporter protein.

RNA Sequencing

Toehold switch sensors are based on synthetic biology. Essentially, they are RNA molecules which code for a reporter protein. They consist of a specific toehold sequence, a ribosome-binding site (which is important for the production of proteins) and a sequence for the reporter protein. The reporter protein can only be produced if the sensor has bonded with its specific target RNA sequence [4]. When producing the sensors, we can select both the toehold sequence and the reporter protein with complete flexibility to match our needs. In this way we can fashion our Wormspotter so that it only sends a desired signal when it binds to RNA molecules specific to T. solium. We are planning to use T. solium-specific RNA sequences for the toehold sequence and beta-Galactosidase as a reporter protein.

Extension PCR

Goal:Assembling our toehold sensors: combining the hairpin-region with a LacZ reporter element

DescriptionEach sensor molecule should consist of the core sensor (recognition structure, characteristic hairpin, ribosome binding site and linker domain), a LacZ reporter element and a T7 promoter region.

1. In a first step, the core sensor was amplified from a circular DNA template.
2. n two PCR-based steps, the recognition and hairpin region was attached to a LacZ reporter element, using primers specifically designed for each individual sensor.
3. In a third PCR step, the sensors were purified and a t7 promoter region was added.

Nested PCR

Goal:Combining two PCR steps into one PCR cycle (increased time-efficiency)

DescriptionAs mentioned above, the assembly of the RNA sensors via extension PCR usually requires two PCR steps in order to amplify the core sensor and furnish it with a LacZ reporter element. The approach of nested PCR can reduce this to one PCR cycle, reducing time and the amount of non-specific PCR products. The approach of nested PCR consists of two processes:

1. In a first run of PCR, the DNA is amplified with a first set of primers. As alternative/similar primer binding sites cannot be fully excluded, a fraction of PCR product will be non-specific.
2. In a second step, the product from the first reaction undergoes a second PCR with a second set of primers. The likelihood of any unspecific product containing binding sites for this second primer set is low, reducing overall contamination.
3. In a third PCR step, the sensors were purified and a t7 promoter region was added.

Colony PCR

Goal:Determine which transformed colony contains the plasmid with the correctly integrated insert.

DescriptionA colony-PCR is a variant of PCR used to directly amplify specific regions of vector DNA from bacteria without having to extract and clean the DNA beforehand. Thus, instead of adding a clean template DNA strand to the PCR reaction mix, whole bacteria from the colonies in question are used. This requires the following steps

1. Primers are chosen in a way that ensures a PCR product containing both vector- and insert-sequences. This allows for monitoring for inverted inserts by checking the size of the PCR product.
2. Colonies are picked with a pipet tip and transferred into purified water, where they get osmotically lysed – this step ensures that the bacterial cell wall is broken and genetic material is accessible
3. Then, PCR master mix is added to the bacteria lysate
4. Finally, PCR is performed in a thermocycler

Gel electrophoresis

Goal:Evaluating the result of a PCR

DescriptionIn order to characterize the product of a PCR, gel electrophoresis can be used to determine the length of the resulting DNA fragment. This is achieved by the following steps:

1. An agarose gel is prepared.
2. The PCR-Product is mixed with a dye and inserted into “pockets” of the gel, along with a “ladder” containing DNA fragments of known size
3. By applying an electrical field, the negatively charged DNA is moved through the gel matrix, separating the DNA fragments by length.
4. The DNA bands are then compared to the ladder and their size can be calculated. By comparing the observed length to the expected length of the fragment, the success of the PCR can be evaluated

DNA Clean Up

Goal:Removing excess nucleotides, salts and additives.

Description