Team:Nevada/Results/

The goal of this project was to generate a number of different nutrient binding proteins for rice. In order to achieve this, the starch binding domain coded by the CBM21 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.

Contents 1 Red Fluorescent Protein-Starch Binding Protein Results 2 Starch Binding Protein-Vitamin B12 Binding Protein Results 3 Starch Binding Protein-Lysine Rich Protein Results 4 Starch Binding Protein-Thiamine Binding Protein Results

Red Fluorescent Protein-Starch Binding Protein Results

In order to demonstrate that the starch binding domain can bind to polished white rice, a new construct was made consisting of a starch-binding domain (BBa_931000) with a red fluorescent domain encoded by the mRFP1 gene (BBa_J23117). This fusion protein construct binds to starch and fluoresces at 607nm. The protein was incubated with rice for five hours and was shown to remain bound even after five water rinses and running the grains under tap water for 30 seconds.
After washing, the rice was examined under a fluorescent microscope for the presence of red fluorescent protein. As the picture below shows, with the rice on the left being a control and the protein treated rice on the right, red fluorescent protein was present in high quantities. Its presence after the washing demonstrates the effectiveness of the starch-binding domain.

With observation and the fluorescent microscope showing the effectiveness of the RFP and of the SBP, it has been confirmed the entirety of the protein functions. This model system provides strong evidence of the success of our iRICE concept and the starch-binding component of our system.

Starch Binding Protein-Vitamin B12 Binding Protein Results

Starch binding protein 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 composite part 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 composite part. After running an SDS-PAGE and staining with Coomassie Brilliant Blue, one band appears representing a pure Starch binding-B12 binding protein. This was further analyzed through a Western Blot analysis. The results of the Western Blot are shown above. 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 graph and accompanying photo above represents the B12 binding assay carried out to show the functioning B12 binding domain of this new brick. SBP-B12 binding protein was aliquoted across 20, 200 ul wells of a binding plate. The protein was left to bind the wells overnight at 4 ͦC. Blocking buffer was added to the wells where the protein had coated, as well 20 wells with no protein coating. The empty wells acted as a control to discredit any non-specific binding of vitamin B12 substrate to the wells or the blocking buffer. Vitamin B12 marked with HRP was added in a decreasing amount to the wells and incubated overnight at 4 C. TMB Microwell Peroxidase substrate (KPL) was added following a wash and allowed to develop. Using Basic Endpoint software, the absorbance at 450 nm was measured for all the wells. The graph represents the numerical results while the visual results are apparent in the picture. The control saw no increase in development and maintained a steady absorbance through all concentrations of vitamin B12 substrate while the SBP-B12 protein showed an initial strong absorbance with a steady decrease as the concentration of substrate decreased. What this demonstrates is the binding capacity of the SBP-B12 protein for vitamin B12. The decrease in absorbance was proportional to the decrease in the B12 substrate which was expected. These results, along with the initial purification of the SBP-B12 protein with an amylose column, theoretically prove the capability of this brick to bind a starch substrate, rice, while simultaneously bridging vitamin B12 to the starch. This also shows that the amount of B12 to be added to a fortification effort utilizing this protein can be controlled. This is an essential point to this project. It is shown that controlled fortification through starch binding proteins is possible.

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.

Starch Binding Protein-Lysine Rich Protein Results

The starch-binding domain (SBP) was conjugated with the lysine-rich protein (LRP) gene derived from C. frutescens to form the new construct, SBP-LRP. A variety of assays and protein purification methods were attempted to show that SBP-LRP would help the lysine-rich protein bind to the rice. Ultimately, the Coomassie stain analysis showed that only the LRP was expressed and the starch binding domain was missing on the protein.

The engineered protein was expressed in E. coli and purified using a Ni-NTA (Thermo Scientific)column. We added 6-his tags to the C-terminal of the lysine-rich protein to simplify the purification of SBP-LRP protein. The Ni-NTA column binds the SBP-LRP protein by interacting with the 6-his tag. The SDS-PAGE stained with Coomassie Brilliant Blue showed one band that represented a pure lysine rich protein as shown above.

Comparing the results from the Coomassie stain and Western Blot showed a successful purification and expression of the LRP protein.

Starch Binding Protein-Thiamine Binding Protein Results

Starch binding protein gene (SBP) was placed at the 5’ end of the thiamine binding protein gene (TBP). The gel below represents digests used to confirm that the SBP-TBP intermediate was successfully constructed (lane 5). The new SBP-TBP gene was digested by Eco I and Pst I resulting in the expected 1300bp product. The SBP-TBP gene was constructed using the thiamine binding protein gene digested by Xba I and Pst I restriction enzymes. The starch binding protein gene was digested with Spe I and Pst I to allow the TBP insert to ligate after it.