Team:Nevada/Results/

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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.

Our next step was to include a positive control, treating the rice with RFP that was not fused to a binding protein. A spraying method was used to treat rice samples with water, RFP, or RFP-SBP. The rice was sprayed 3 times for approximately 1 second per spray with an aerosol sprayer normally used for TLC plates. The rice was then left alone to dry for 3 hours and then washed with two traditional methods, pinching and swirling, as well as placed under the tap for about 15 seconds. This overall process represents treatment at the factory(spraying), buying dry rice from the store, and home washing before cooking.

Binding Tests: An aerosol sprayer was used to treat rice with one of three treatments. The treatments were water, RFP, and RFP-SBP. The rice was sprayed three times for one second then dried for three hours. The rice was subsequently washed through the swirling method, the pinching method, and it was placed under the tap for about 15 seconds. The rice was analyzed under a fluorescent microscope in the first picture and under white light in the second picture.

The image above is of a slide with three grains of rice. One is treated with water, one is treated with RFP, one is treated RFP-SBP. The concentration of RFP with the RFP-SBP treated rice is significantly higher than in the other two samples. The presence of RFP on the rice, though in lower amounts, suggests that starch, when wet, is an adequate binder of protein likely due to its hydrophobic nature. This could mean that binding proteins might be usable without fusing them to starch-binding protein. Although, binding was significantly more successful with starch-binding protein.

With observations 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 encoded by the CBM 21 gene derived from E. coli was conjugated with the BtuF gene derived from a membrane transporter system used by E. coli in B12 transport. Through various assays and protein purification methods, the functionality of both domains of this new composite part has been demonstrated.

In order to purify the engineered protein after it was expressed by E. coli, an amylose column (Thermo Scientific) was used to selectively bind the starch-binding domain of this protein. Procedurally and functionally, this column works similar to a Ni-column in that the Starch binding domain acts similar to the “his” tags on proteins used in Ni-column purification. The amylose column also demonstrates the binding abilities of the starch-binding domain of this composite part. After running an SDS-PAGE and staining with Coomassie Brilliant Blue, a band appears in lane 1 which represents the Starch binding-B12 binding protein. This was further analyzed through Western Blot analysis. The results of the Western Blot are shown above.In lanes 9 and 10, which represent the crude and purified protein extract from E. coli respectively, strong bands appear. There are other bands present in the lanes, but after further research, it was found that this is a characteristic of the Btuf gene. In a paper by Nathalie Cadieux et al, the researchers demonstrated through a similar Western Blot analysis that there are two forms of the B12 binding domain once expressed. One is a mature protein, and the other an immature and slightly larger protein form of the same B12 binding protein. After comparing the Western blot and Coomassie stain, it was concluded that the purification was successful. By purifying SBP-B12 binding protein through this method the functionality of the starch binding domain was demonstrated.

The graph and accompanying photo above represent the B12 binding assay. Starch binding-B12 binding protein was aliquoted across 20, 200 ul wells of a binding plate. The protein bound to the wells overnight at 4 ͦC. Blocking buffer was added to the wells where the protein had coated, as well 20 wells with no Starch binding-B12 bind protein coating. The empty wells acted as a control to show that there was not 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 rigorous wash and allowed to develop. Using Basic Endpoint software, the absorbance at 450 nm was measured for all the wells. The above graph represents the quantitative results of this assay while the qualitative results are shown in the above picture. The control saw no increase in development and maintained a steady absorbance through all concentrations of vitamin B12 substrate. The SBP-B12 protein showed an initial strong absorbance with a steady decrease as the concentration of substrate decreased. This demonstrates the binding ability 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 protein to bind a starch substrate, rice, while simultaneously bridging vitamin B12 to the starch. Although amylose was used, the starch binging domain has been shown to show equal, if not greater, binding affinity to numerous carbohydrate rich substrates. This includes rice starch, corn starch, potato starch, and various others. This also shows that the amount of B12 added in a fortification effort utilizing this protein can be controlled. This is an essential point to the project.

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 column (Thermo Scientific). 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.