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

High throughput screening in liquid medium

Goal:High-throughput screening for sensors that react specifically to target RNA

DescriptionIn silico design and PCR allows for assembly of a large number of potential sensor candidates. In order to evaluate which of these sensors reacts to the target RNA in a sensitive and specific manner, the following steps are required:

1. A master mix of cell free expression system, reporter molecule and certain amount of target RNA or control RNA is prepared and transferred to a special low-volume 384-well plate.
2. Adding sensor DNA to each well
3. Measure absorption at 405nm (yellow – starting color) and 560nm (violet – end color) in a GloMax Discover ® plate reader (Promega) for 2-4 hours at 37° C in order to follow the kinetic of the reaction from yellow to violet.
4. Results files are evaluated using a python script. By automating the process of data evaluation, a empirically sound conclusion can be reached in less than 15 minutes.

Reliable cell free expression on cellulose membranes

Goal:Membranes are known to be efficient microfluidic environments, but due to the charge of nucleic acids and the negative charge of cellulose, optimization is required to make the system storable and the color change easily recognizable.

DescriptionIn silico design and PCR allows for assembly of a large number of potential sensor candidates. In order to evaluate which of these sensors reacts to the target RNA in a sensitive and specific manner, the following steps are required:

• Blocking the charge of the cellulose membrane.
  Classic approaches of membrane blocking like incubation with 5% BSA or milk powder failed due to negative interaction with the cell free expression system. Hence we used an amphiphilic component, creating a liquid barrier shielding the sensor system from negative charges and successfully creating a stable color reaction on the membrane.
• Altering the composition of our sensor system components, to maximize color reaction.
  We discovered that about one third of color intensity is lost on membranes in comparison to fluid environments. Reactions on membranes thus require to double the amount of sensor molecule added to the reaction
• Stabilizing the cell-free system for storage by lyophilisation
  We succeeded in freeze-drying the cell-free expression system onto cellulose membrane and were furthermore able to trigger an active color change after resuspension.

Goal:Establishing the PureExpress® cell free protein expression kits (PureExpress®, NEB) as our main tool for RNA detection and assess its capabilities.

DescriptionThe PureExpress®-System is composition of completely recombinant molecules to effectively transcribe any form of DNA into mRNA and then translate this into a protein. To use this system for field diagnostics, it has to meet the following requirements:

• Heat stability up to a temperature of 50°C
• High efficiency even with low volumes of reagents and/or low concentrations
• Stability towards changes in electrolyte concentration/ addition of foreign molecules
• Long-term storability.

We verified this by evaluating the kinetics of sensor reactions in different experimental conditions, including different temperatures, different reactant concentrations, changes in buffer as well as re-measurements after storage.

We could show that the system meets all these requirements. Especially noteworthy, we were able to create significant color change in a volume of 2,5 uL which is only 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 use the system with patient samples of only estimated concentration without risking reduced functionality. Last but not least, the system is not vulnerable to possibly interfering molecules, as for example the chlor-phenol-red used as a reporter dye.

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

Cloning into pSB1C