Team:Nevada/Results/

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Revision as of 23:54, 3 October 2012

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 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 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.

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-	The graph above represents the B12 binding assay carried out to show the functioning B12 binding domain of this new brick ~~which is visually represented by the accompanying picture~~. ~~Purified~~ 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. ~~After rinsing each well, 5% blocking~~ buffer ~~made with PBS-T and non-fat milk powder~~ 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 ~~disprove~~ any non-specific binding of B12 substrate to the wells or the blocking buffer. Vitamin B12 marked with HRP was added in a decreasing ~~manner across all~~ the wells and ~~then allowed to interact with the protein coat~~ overnight at 4 C. TMB microwell peroxidase substrate was added following a wash and allowed to develop. Using Basic Endpoint software, the absorbance at 450 nm was measured for all the wells. ~~This~~ graph represents the results. The control saw no increase 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.	+	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 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.		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.