Difference between revisions of "Part:BBa K2804005"
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===Usage and Biology===
 
===Usage and Biology===
  
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<p align="justify">The Microbial Fuel Cell (MFC) can be a future environmentally friendly biotechnological application for the production of electrical energy. As a future alternative energy source, the bioelectricity generation must become more efficient. A major limiting factor is the low bacterial membrane permeability, hindering transport of electron shuttles through the membrane and thereby restricting the electron shuttle-mediated extracellular electron transfer (EET) from bacteria to electrodes. This results in a reduced electrical power output of the MFC. Therefore, we heterologously expressed the porin protein OprF from ''Pseudomonas fluorescens'' into ''Escherichia coli''.</p>
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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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[[<img src="https://static.igem.org/mediawiki/2018/e/e4/T--Ecuador--CBD-sfGFP.png">'''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--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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===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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[[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

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