Difference between revisions of "Part:BBa K2804005"

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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>]]
 
[[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===
 +
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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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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[[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--panchoagua.png|400px|center|thumb|<p align="justify">'''Figure 1: Schematic overview of the enhancement mechanism of electron shuttle-mediated electron transfer between bacteria and the anode of MFCs by the synthetic porin OprF. Oxidized mediators diffuse into the periplasmatic space where they accept electrons. Reduced mediators are secreted through outer membrane porins and donate their electrons to the electrode.'''</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).
  
 
===References===
 
===References===
  
 
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.
 
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.

Revision as of 02:11, 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. 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.

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

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.