Part Collection

Part Collection: Synthetic Biology Detects Defective Maple Syrup


Our collection of parts is designed to be used in tandem for the detection of defective “buddy” maple syrup.

The Problem


The annual maple sugaring season begins in late winter and ends in early spring, lasting only until the trees begin to grow buds. At the time of bud development, the trees undergo metabolic changes, resulting in a slightly altered composition of the sap. When this altered sap is collected and boiled down into syrup, chemical reactions occur resulting in a defective cabbage-like taste and odor. This defective syrup, termed “buddy” syrup, is deemed not suitable for human consumption and is discarded, resulting in significant losses in profits. Currently, this annual defect can only be detected once the sap has been processed to syrup, resulting in a loss of time and fossil fuels.


Solution


Team Sapatasense has developed a toolbox of techniques to detect “buddiness” in maple sap straight from the tree, maximizing farmer’s profits and minimizing waste. Our toolbox features a whole-cell bacterial biosensor for asparagine levels, an aptameric biosensor for sarcosine, and an enzymatic biosensor for choline. These three novel biosensors work together for the accurate detection of defective maple sap.

The chemical composition of “buddy” sap is characterized by an increase in the amino acid asparagine. Therefore, we chose to leverage this as a biomarker for the detection of the defect.
To detect asparagine, we employed our novel unival whole-cell biosensor, capable of detecting nearly any compound. To impart specificity towards asparagine, we incubated our genetically engineered bacterial strain with an anti-asparagine antibody. Our results demonstrate that our biosensor is capable of distinguishing between asparagine concentrations.

How it works:
Part BBa_K4130000 codes for a protein known as EibD. EibD is an outer-membrane protein capable of binding non-immunologically to the constant region of antibodies. Thus, expression of EibD to the surface of bacteria, followed by incubation with an antibody, results in antibody-coated bacteria. Antibody-coated bacteria can then bind the antigen of interest, producing a quantifiable signal.
The read-out of our novel assay is “autoaggregation” or bacterial clumping. Upon expression of EibD, the bacteria clump together due to homophilic EibD-EibD interactions. This response can be quantifiably measured via a sedimentation assay. Upon incubation with the antibody, homophilic EibD-EibD interactions are replaced by EibD-antibody interactions, resulting in a reduction of autoaggregation. Finally, addition of the antigen results in a further reduction in autoaggregation, likely due to antibody-antigen interactions reducing the presence of antibody-antibody interactions.
This universal assay was applied to the detection of asparagine by incubation with an asparagine-specific antibody.
Defective “buddy” sap is also characterized by an increase in sarcosine concentrations. Thus, we developed an aptameric biosensor for the detection of sarcosine.

How it works:
Aptamers are short sequences of DNA or RNA that exhibit affinity toward specific molecules, taking on a 3D conformation upon binding to its target. Our DNA aptamer, coded by Part BBa_K1340014, has been previously shown to have high affinity and specificity towards the small molecule sarcosine. We attached our aptamer to cost-effective, miniaturized carbon electrodes using a mixture of chitosan and reduced graphene oxide. Methylene blue was also added to improve sensitivity of the system.
Upon addition of the target molecule, sarcosine, the aptamers shift 3D conformation and bind sarcosine close to the electrode surface, reducing the flow of electrons, increasing the resistance. Therefore, resistance was used as the read-out of this assay.

As sap becomes more “buddy” there is a concomitant increase in choline concentration. Therefore, we also developed a bioassay to differentiate between concentrations of choline in maple sap.

How it works:
Part BBa_K130005 codes for a protein known as “choline oxidase”, capable of catalyzing the conversion of choline to glycine betaine, producing hydrogen peroxide as a byproduct. As choline concentrations are increased, choline oxidase produces more hydrogen peroxide.
To develop our biosensor, we used the BioBrick BBa_K130005 to express and then purify choline oxidase. Purified choline oxidase can then be used in a 96 well-plate assay, where choline substrate is added and hydrogen peroxide is produced in amounts proportional to choline. Hydrogen peroxide concentration can then be measured in a colorimetric assay, and related back to the original choline concentration in the sample.


Parts as a Collection


Early on in our project, we realized that generating a precise prediction of “buddiness” utilizing only one biomarker would be nearly impossible. Inter-tree variability and sporadic changes in weather and temperature conditions produce unavoidable noise when collecting a single sap sample. Therefore, it is critical that multiple biosensors must be integrated to predict one outcome: “buddiness”. Our work utilizing parts BBa_K130000, BBa_K130014, and BBa_K130005 should thus be combined and viewed as a functional “part collection” for the detection of defective maple sap.