Team:Nevada/Results/

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(Starch Binding Protein-Vitamin B12 Binding Protein 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.
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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.
==Red Fluorescent Protein-Starch Binding Protein Results==
==Red Fluorescent Protein-Starch Binding Protein Results==
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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.
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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.
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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.
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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.
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<html><center><img src="https://static.igem.org/mediawiki/igem.org/6/68/RFP_glowing.jpg"> </img></center></html>
 
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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.
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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 than 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.
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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.  
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==Starch Binding Protein-Vitamin B12 Binding Protein Results==
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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.  
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<html><center><img src="https://static.igem.org/mediawiki/2012/4/47/Revised_2.jpg"> </img></center></html>
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<b>Binding Test:</b> 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.
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<html><center><img src="https://static.igem.org/mediawiki/2012/d/d0/B12-Results-1.jpg"> </img><img src="https://static.igem.org/mediawiki/2012/1/19/Justin%27s_Blot.JPG"> </img> </center> </html>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.  
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==Starch Binding Protein-Vitamin B12 Binding Protein Results==
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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.  
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<html><center><img src="https://static.igem.org/mediawiki/2012/d/d0/B12-Results-1.jpg"> </img><img src="https://static.igem.org/mediawiki/2012/1/19/Justin%27s_Blot.JPG"> </img> </center> </html>
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<html><center><img src="https://static.igem.org/mediawiki/2012/0/0a/B12-Results-2.jpg"> </img> <img src="https://static.igem.org/mediawiki/2012/d/d2/B12-Results-3.jpg"></img></center></html>
 
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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.
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<html><center><img src="https://static.igem.org/mediawiki/2012/0/0a/B12-Results-2.jpg"> </img> <img src="https://static.igem.org/mediawiki/2012/d/d2/B12-Results-3.jpg"></img></center></html>
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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.
 
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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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==Starch Binding Protein-Lysine Rich Protein Results==
 
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The starch-binding domain was engineered into the lysine-rich protein 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 (LRP) bind to the rice. Ultimately, the Coomassie gel results showed that only the LRP was expressed and the starch binding domain was missing on the protein. 
 
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<html><center><img src="https://static.igem.org/mediawiki/2012/4/4a/Michelle1.JPG"> </img></center></html>
 
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The engineered protein was expressed in E. coli and purified using a Ni-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-column binds the SBP-LRP protein by interacting with the 6-his tag tail. The SDS-PAGE by Coomassie Brilliant Blue staining showed one band that represented a pure lysine rich protein as shown above.  
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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.
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<html><center><img src="https://static.igem.org/mediawiki/igem.org/1/1c/Michelle2.JPG"></center> </img></html>
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==Starch Binding Protein-Thiamine Binding Protein Results==
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Comparing the results from the Coomassie stain and Western Blot showed a successful purification and expression of the LRP protein.
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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.  
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==Starch Binding Protein-Thiamine-Binding Protein Results==
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<html><center><img src="https://static.igem.org/mediawiki/igem.org/d/dd/Chris.jpg"> </img></center></html>

Latest revision as of 04:01, 27 October 2012



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 than 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.