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Overview

Prior CRISPR transcriptional factors

CRISPR transcriptional factors are a breakthrough because they enable control of the expression of a particular gene – based on the gRNA. In these systems, the gRNA is attached to a CAS (CRISPR associated) protein, often CAS9. These proteins are degraded so that they do not cleave the dsDNA. The CAS9 protein is attached to either a repressor or an enhancer, which modulates the expression of the gene. Ubiquitination enables a system that can change only once. CRISPR transcriptional factors were first developed by Perez-Pinera et al. in 2013.

Auxin Background

Auxin-IAA is a plant hormone that signals the development of leaves. This molecule serves as a model molecule for detection by the CRISPR transcriptional factors because it can pass through the membrane and because it has well-characterized corresponding F-box (AFB2) and degron (deg1). IAA is used in almost all plants, and is created by the from the amino acid tryptophan, and the synthesis is well characterized.

Design:

The pathway relies on CRISPR transcription factors to produce an indigo response to the plant hormone auxin-iaa (indole-3 acetic acid).

The Cas9 with a deactivated nuclease, or dCas9, binds to a gRNA strand that is complementary to the region of S. Cerivisiae that is near the inserted lacZ gene. Mxi1 is a repressor protein. In the presence of auxin, the degron protein recruits an F-box and a ubiquitin ligase (2). This E3 ubiquitin ligase tags the protein complex for degredation.

The β-galactosidase enzyme protein (3) catalyzes the separation of X-gal into galactose and 5-bromo-4-chloro-3-hydroxyindole. The 5-bromo-4-chloro-3-hydroxyindole then dimerizes and is oxidized to form the visible blue color.

The CRISPR transcriptional factor is an optimal method for sensing molecules because the components can be optimized and substituted. In this case, the AFB2 protein serves as the F-box and the Deg1 protein serves as the degron. Also, this system is applicable to other genes and organisms with dsDNA.

Test Strip Design

The design for the yeast biosensor needs to create an ideal environment for the culture.

The base of the test strip, chromatography paper, The PDMS is ideal for a test strip because it is manufactured rapidly at a low cost. The PDMS window allows small molecule-gasses to permeate but not foreign contaminants. The one-way valve would most likely have a connection to a small pipette that could deposit medium evenly across the yeast cells. The chromatography paper spreads the test solution evenly so that different sections of the yeast media have the same concentration of test solution. In this way, the indigo color will be even and predictable in the yeast section. A section of the strip will have yeast that constitutively expresses indigo as a control to ensure that the rehydration is functional.

Lateral Flow Test Strip Background

In a typical lateral flow assay, there are enzymes in the test strip that are in a dried salt and sugar mix.

While the sample fluid dissolves the salt-sugar matrix, it dissolves the particles and the sample and enzymes and salt and sugar mix. The analyte binds to the particles while traveling to the third capillary bed. This material has one or more areas (often stripes) where a third molecule has been placed by the manufacturer. By the time the sample mix reaches these strips, the analyte has been bound to the enzyme and the third 'capture' molecule binds the complex, and changes color. The color increases as enzyme-analyte-third molecules accumulate.

Saccharomyces Cerivisiae Background

The type of cell that was engineered is Saccharomyces Cerivisiae, commonly known as baker’s yeast. This cell has an activity > 0 for pH from 2.1 to 7 (Arroyo et al. 2009). The activity is above 0 and increasing from 12°C to 36°C (Arroyo et al. 2009). The wild type Saccharomyces Cerivisiae is not known to be mutagenic. Another crucial characterization for test strip media is longevity. S. Cerivisiae can live for approximately 20 to 120 hours (Minois et al. 2004). In a dehydrated dormant state, however, the yeast can survive for years (Fabrizio & Longo 2003). The genome of this model organism has been sequenced, it is easy to obtain in the lab, and the genomic structure is easy to modify.