Team:Nevada/Results/

The goal of this project was to provide a method for bridging a variety of nutrients to rice. In order to achieve this, the starch binding domain coded by the CBM 21 gene from R. oryzae was conjugated with various nutrient binding proteins. We believe this design to be sound based on some very promising qualitative results obtained from the RFP-SBP construct, as well as quantitative results obtained from the SBP-B12BP construct.

RFP-SBP Results

SBP-B12BP Results

SBP was conjugated with the BtuF gene from the membrane transporter used by E. coli in B12 transport. Through various assays and protein purification methods, the functionality of both domains of this new brick have been shown to work effectively.

In order to purify the engineered protein after it was expressed by E. coli, an amylose column was used to selectively bind the starch binding domain of this protein. In a sense, this column works similar to a Ni-column in that the SBP domain acts similar to the “his” tags on proteins used in Ni column purification. The amylose column also serves as way of showing the binding efficiency of the starch binding domain of this new brick. After running an SDS-PAGE and staining with Coomassie Brilliant Blue, one band appears representing a pure SBP-B12 binding protein. This was further analyzed through a Western Blot analysis. After comparing the Western blot and Coomassie stain, it was concluded that the purification was successful. By successfully purifying the protein through this method the functionality of the starch binding domain was demonstrated.

The binding efficiency of the B12 binding domain of this brick was analyzed through a different type of binding assay. A 96-well binding plate typically used for ELISA was coated with the SBP-B12 binding protein. B12 conjugated with Horseradish Peroxidase (HRP) was added in a decreasing amount to the wells and was then washed numerous times. TMB Microwell Peroxidase substrate was added to develop the HRP. Spectrophotometric data was obtained to further prove the qualitative data obtained from this assay. The results are represented in graphical form above. The highest concentration of B12-HRP developed the strongest and the development decreased proportionally to the decrease in concentration. A control was used to show that there was little to no non-specific binding of the B12-HRP to either the well itself or to the blocking buffer. According to the results there is very little non-specific binding which further enhances the results of the binding assay.

With both the amylose column purification and the B12 binding assay, it can be concluded that both domains of this new brick function. Amylose is just one substrate of the SBP domain and there are numerous others. The SBP domain has been shown to have an even higher affinity for rice starch, therefore, theoretically this new brick will effectively bind to rice and, with the B12 binding domain, bridge Vitamin B12 to the rice.