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.

Red Fluorescent Protein-Starch Binding Protein Results

To demonstrate that the starch binding protein domain (SBP) will bind to polished rice grains, a fusion construct was made consisting of the starch-binding domain (SBP)(BBa_931000) from the Rhizopus oryzae amylose gene and red fluorescent protein (RFP)(BBa_J23117). The resulting construct was expressed in E. coli behind a constitutive promoter to produce the RFP-SBP protein.

To assess the ability of RFP-SBP to bind rice, dry rice grains were sprayed with a dilute crude extract made from the RFP-SBP expressing bacteria. The rice was dried for one hour and then washed for 15 minutes with copius amounts of water. The presence of the RFP-SBP protein on treated rice grains was then determined by fluorescent microscopy. The results of this experiment showed the RFP-SBP treated rice fluoresced strongly, while untreated rice (i.e. rice first sprayed with water only)did not fluoresce at all.

To address the possibility that RFP-SBP binding to rice was non-specific, rice was also treated with a crude extract prepared from E. coli expressing RFP alone. To standardize the treatment, the RFP crude extract was diluted so its relative fluorescence was equivalent to the SBP-RFP crude extract. The results from this experiment showed that while RFP treated rice fluoresced stronger than the untreated water control, the signal was clearly weaker that that seen from the RFP-SBP treatment. This suggest that while the SBP domain does direct binding of RFP to rice, a fair amount of non-specific binding also occurs due to the hydroscopic nature of starch.

While SBP appears to promote better and more specific binding to rice, because of rice’s inherent high protein binding capacity, an alternative approach to SBP fusion proteins would be to bind nutrients to rice using the nutrient binding proteins alone. However, because the amino acid sequence of each protein is unique, the relative binding strength to rice could not be predicted. Therefore, the use of SBP fusion would provide more reliable and stronger binding to rice.

Binding Test: An atomizer was used to spray rice with the following treatments: 1) water; 2) crude E. coli extract containign RFP; and 3) crude E. coli extract containing RFP-SBP. The rice was sprayed in 3 one second burst and then allowed to 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 then analyzed under a fluorescent microscope (excitation 584 nm, emmission 607nm) (top photo). Lower photo shows white light image of same samples.

Starch Binding Protein-Vitamin B12 Binding Protein Results

Starch binding protein (SBP) was fused to the Vitamin B12 Binding protein encoded by the "E. coli" BtuF gene. The BtuF protein is a periplasmic protein involved in the Vitamin B12 ABC transporter system used by E. coli to scavenge Vitamin B12 from its environment. 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 and reconfirms our findings from the RFP-SBP studies described above. 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 during expression. One is a mature protein, and the other is an immature and slightly larger 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-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.