Difference between revisions of "Part:BBa K2804005"
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<partinfo>BBa_K2804005 short</partinfo>
 
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The superfolder green fluorescent protein (sfGFP) is fused to the C-terminus of the cellulose binding domain (CBD) of the scaffolding protein cipA of Clostridium thermocellum.
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===Usage and Biology===
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<p align="justify"> Cellulose binding domains (CBDs) have been widely used in scientific research for their biotechnological applications. Originaly, they are part of the more efficient cellulases improving the binding of the catalytic domain.<sup>1</sup> In this part, the CBD of the scaffolding protein cipA of one of the most efficient cellulolityc bacteria, "Clostridium thermocellum", is fused to sfGFP with a 6X Histidine tail in order to visualize and measure at single-molecule level the interaction between the CBD cipA and bacterial cellulose.<sup>2</sup></p>
  
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[[Image:T--Ecuador--CBD-sfGFP.png|400px|center|thumb|<p align="justify">'''Figure 1: Tridimensional structure of CBD cipA-sfGFP obtained from the server I-Tasser.'''</p>]]
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===Usage and Biology===
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===Experiment===
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Measurements of remaining fluorescences, after different washes, were done according to the protocol described by the Team Imperial 2014 (http://2014.igem.org/Team:Imperial/Protocols). Briefly, 20g of kombucha cellulose were blended with 125 mL. Then, 100 uL of the mixture were added into each well of a 96-plate reader and dried at 60º for 5.5 hours using a  stove. A volume of 100 uL of CBD cipA-sfGFP protein extract was added into each well an incubated at 4ºC overnight. The fluorescence was measured after washing with different substances: dH20 and 70% EtOH.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2804005 SequenceAndFeatures</partinfo>
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[[Image:T--Ecuador--panchoagua.png|400px|center|thumb|<p align="justify">'''Figure 1: Remaining fluorescences after three washes with dH20.'''</p>]]
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[[Image:T--Ecuador--panchoetol.png|400px|center|thumb|<p align="justify">'''Figure 2:  Remaining fluorescences after three washes with 70%EtOH.'''</p>]]
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====Conclusion====
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From the figures we can conclude the CBD cipA provides a binding stregth higher than the negative control GFP (p value≤0.01).
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===References===
  
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1. Kruus, K., Lua, A. C., Demain, A. L., & Wu, J. H. (1995). The anchorage function of CipA (CelL), a scaffolding protein of the Clostridium thermocellum cellulosome. Proceedings of the National Academy of Sciences of the United States of America, 92(20), 9254–9258.
  
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2. Zhang, M., Sheng-Cheng, W., Zhou, W. & Xu, B. (2012).Imaging and Measuring Single-Molecule Interaction between a Carbohydrate-Binding Module and Natural Plant Cell Wall Cellulose. J. Phys. Chem. B, 2012, 116 (33), pp 9949–9956
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===Functional Parameters===
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<partinfo>BBa_K2804005 parameters</partinfo>
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Latest revision as of 02:18, 18 October 2018


CBD cipA fused to sfGFP under the control of LacI promoter

Usage and Biology

Cellulose binding domains (CBDs) have been widely used in scientific research for their biotechnological applications. Originaly, they are part of the more efficient cellulases improving the binding of the catalytic domain.1 In this part, the CBD of the scaffolding protein cipA of one of the most efficient cellulolityc bacteria, "Clostridium thermocellum", is fused to sfGFP with a 6X Histidine tail in order to visualize and measure at single-molecule level the interaction between the CBD cipA and bacterial cellulose.2

Figure 1: Tridimensional structure of CBD cipA-sfGFP obtained from the server I-Tasser.

Experiment

Measurements of remaining fluorescences, after different washes, were done according to the protocol described by the Team Imperial 2014 (http://2014.igem.org/Team:Imperial/Protocols). Briefly, 20g of kombucha cellulose were blended with 125 mL. Then, 100 uL of the mixture were added into each well of a 96-plate reader and dried at 60º for 5.5 hours using a stove. A volume of 100 uL of CBD cipA-sfGFP protein extract was added into each well an incubated at 4ºC overnight. The fluorescence was measured after washing with different substances: dH20 and 70% EtOH.

Figure 1: Remaining fluorescences after three washes with dH20.

Figure 2: Remaining fluorescences after three washes with 70%EtOH.

Conclusion

From the figures we can conclude the CBD cipA provides a binding stregth higher than the negative control GFP (p value≤0.01).

References

1. Kruus, K., Lua, A. C., Demain, A. L., & Wu, J. H. (1995). The anchorage function of CipA (CelL), a scaffolding protein of the Clostridium thermocellum cellulosome. Proceedings of the National Academy of Sciences of the United States of America, 92(20), 9254–9258.

2. Zhang, M., Sheng-Cheng, W., Zhou, W. & Xu, B. (2012).Imaging and Measuring Single-Molecule Interaction between a Carbohydrate-Binding Module and Natural Plant Cell Wall Cellulose. J. Phys. Chem. B, 2012, 116 (33), pp 9949–9956