Team:Washington/Auxin

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Introduction: Auxin-IAA Pathway

Prior CRISPR transcriptional factors

CRISPR transcriptional factors enable scientists to make targeted changes in gene expression through the use of gRNA. The gRNA attaches to the CRISPR associated protein, and guide it to a specific DNA locus based on the sequence of the gRNA. The CRISPR proteins we used were obtained from the Klavins lab, and are designed so that they do not cleave the DNA strands themselves, but will instead bind tightly and prevent access to the gene by other proteins. This effectively disables transcription and translation of the targeted gene. CRISPR transcriptional factors were first developed by Perez-Pinera et al. in 2013.

Auxin is a class of plant hormone that is involved in developmental and behavioral signalling. This type of molecule serves as a good proof-of-concept for the detection of small-molecules by our model system. This is because our Auxin can pass through the cell membrane and bind to an F-box protein (AFB2) and a degron (deg1), which will then target our dCas9 protein for degradation. Indole-3-acetic acid (IAA) is the specific Auxin molecule that we are using in our model system.

Auxin Design:

The pathway relies on several components. Firstly, a dead-CRISPR transcription factor fused to a degron domain and a repressor domain. Along with a guide RNA designed to target and upstream activating site. Combined the dCas9 complex along with the guide RNA will suppress the expression of beta-galactosidase. However, in the presence of IAA and an F-Box protein, the dCas9 complex will be degraded and beta-galactosidase will be expressed. Thus, causing another small molecule X-Gal to be converted into indigo and appear blue, a color response. The system is designed to produce a response (a color response in our case) to the presence of the small molecule indole-3-acetic acid.

(1) dCas9-deg1-MXI1 binds to a guide RNA which targets a sequence of a LacZ promoter causing the expression of beta-galactosidase to be supprefont size = 2ssed. (2) Auxin binds to both AFB2 (which also helps recruit the ubiquitin ligase) and deg1 simultaneously bringing the two into close proximity allowing the ubiquitin ligase to ubiquitinate the dCas9 construct. (3) LacZ expression is no longer suppressed allowing beta galactosidase to be produced and in the presence of x-gal, indigo is then formed.

Results

The transformation of the lacZ gene proved successful. The gene proved unusually uncooperative. Salmon sperm and fresh cultures were an absolute must. Cells usually take at least 30 min to show faint changes in color. Our selection was URA.

The transformation of the blue chromoprotein was a failure. The gene was put in the yeast, but did not express any color. Our selection was URA.

The transformation of the gRNA was mixed. Auxin assays proved that the cells did contain the construct. However, in one experiment, two cell cultures (one with auxin added, one without) both turned blue. The cells with auxin added were significantly more blue. Possible explanations include:

1. The “yeast” was actually bacteria containing the lacZ gene.

2. The digestion prior to transformation was faulty. We observed several mysterious bands when we ran a gel on the digest. Some cells may have therefore only inherited part of the construct. Our selection was HIS, so some cells may have acquired a functional HIS gene, but did not have the gRNA due to the digestion cutting our plasmid in the wrong places.

Future Direction

Troubleshoot guide-RNA (or not)

At this point in time, the cells containing the F-Box, dCas9-degron-inhibitor construct, the guide RNA, and the beta-galactosidase construct convert X-Gal into indigo in the presence of auxin. However, during certain runs of our experiment without auxin, indigo was still formed. We are currently examining this issue.

Introduce high-resolution, easily-quantifiable response gradient using ONPG

ONPG is a molecule used in a liquid assay and can be measured quantitatively with very high-resolution. This works by using a response factor that is able to dissolve in liquid solution and is thus measurable via photospectroscopy. Using this method, our team can precisely measure the impact of varying concentrations of small molecules on our system. We can then use the measured, overall response of our system to predict the amount of analyte present.

Alternatively, use a quicker and easily visible response factor

Currently, the response time of our system utilizing beta-galactosidase to cleave x-gal is somewhat lengthy. By switching over to a rapidly produced, colored signal response that is visible to the naked eye we hope to make the system easier to use. Colored response signals such as RFP are great because they can be visualized without the need for lab instruments.

Find or design a protein similar to AFB2 that can target other toxins/small molecules

The limitations of our model system is that only Auxin like molecules can be detected. However, with future advancements in the field of protein engineering perhaps more complex molecules can be detected using our system.