Homebrewed Diagnostic Enzymes

The printing press democratized books, allowing for the reduction in cost and widespread distribution of books. This made literature accessible to more people by reducing the cost barrier of reading, making reading possible to more people. The impact the printing press made on reading was largely because of scalability - the physical resources and time needed to make a single book were greatly reduced. Now we live in a largely literate world.

Diagnostic testing of viral diseases including COVID19 is not new, and is done using an established laboratory technique (RT-PCR, and more recently LAMP). Books had been written long before the invention of the printing press, but they were expensive. There is a lack of access to RT-PCR reagents (proteins) as they are expensive, the same way books were when they were handwritten. We want to live in a world that is largely tested for COVID19 the way that it is already literate.

In times of infectious disease outbreaks including COVID19, when catching the disease early on is critically important to reducing its spread, diagnostic laboratories need rapid access to proteins. Making the reagents needed to do RT-PCR more affordable reduces the overall cost barrier of diagnostic testing, making testing possible in laboratories with fewer financial resources and in places that are very far from enzyme supply companies. With a lower cost of each diagnostic test, tests can be done more frequently and for more people around the world. More diagnostic testing will give a clearer picture of where the disease is, allowing us to contain it, even before a clinical trial of a vaccine or antiviral drug is complete.

Problem:

There is a lack of access to diagnostic testing for COVID19. Reagents for diagnostic testing are expensive, the most expensive of those reagents are proteins.

Solution:

Producing proteins using a methodology that is affordable and scalable will make diagnostic testing possible in more places by reducing the cost barrier of diagnostic testing. 

Strategy:

By engineering yeast using CRISPR to produce the enzymes needed for diagnostic tests, the engineered yeast can be used instead of conventional brewer's yeast when brewing beer. At the end of the brewing process, when the yeast is separated from the beer and disposed of by the homebrewer or brewery, it can be used in place of the expensive enzymes in the diagnostic procedures.

Project Goals:

1) To express the genes that code for the diagnostic enzymes from chromosomal DNA in yeast by integrating those genes into the yeast genome using CRISPR. 

2) to use that yeast to brew beer

- Nico has access to everything he needs to do this

3) to filter the yeast from that beer

- Nico has access to everything he needs to do this

4) to do PCR reactions using that yeast as a cellular reagent in place of conventional thermostable DNA polymerase from a laboratory supply company in order to test the sensitivity of PCR reactions done using those cellular reagents as a way to detect very small amounts of DNA/RNA for diagnostic procedures. The sensitivity of PCR reactions done using this engineered yeast as the enzyme source can be compared to the sensitivity of conventional PCR master mix by creating a dilution series of template DNA, and running PCR reactions using the dilution series of template DNA. Once it has been established that the DNA polymerase cellular reagent method is sufficiently sensitive to detecting DNA, the same approach will be applied with a reverse transcriptase enzyme coding sequence engineered into yeast using the same approach for cloning, and for testing it's function in RT-PCR reactions. 

- Nico has access to everything he needs to do this other than the engineered yeast.