Biomod/2011/Tianjin:Results: Difference between revisions

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==''' Cell-free synthesis of actin-like cytoskeleton filaments'''==
The expression of the MreB-RFP fusion protein and RFP, both with an N-terminal His6 tag, using the cell-free expression system was verified by SDS-PAGE (Fig 7), indicating that our cassette was successful expressed in vitro. Both MreB and RFP protein are around 30 kDa in size.
[[Image:Biomod tianjin results 01.png|center|600px|thumb|Fig 1.Results of fusion protein MreB-RFP expression in vitro. (A) SDS-PAGE (15%) of His6 tagged RFP and MreB-RFP proteins overexpressed using the cell-free expression system; (B) Reaction solution after the cell-free expression of MreB-RFP. Samples turning to pink can be detected by eye. ]]


=='''Cell-free expression in W/O/W microdroplets'''==
Results from fluorescence micrograph and confocal micrograph supported the compatibility of double emulsions with cell-free expression of soluble and structural membrane-related proteins.
[[Image:Biomod tianjin results 0.png|center|600px|thumb|Fig 2. Cell-free protein expression in W/O/W. Fluorescence micrograph of a W/O/W during the expression of (a) RFP as a soluble protein in the protocell cytosol, and (b) MreB-RFP, which has portioned at the hydrophobic interface. Figure (c) shows a confocal micrograph (3um thick plane) of a W/O/W, 14 h after generation. The RFP-tagged MreB polymerized, forming aggregates localized to the oil-water interface. The image was created by forming the W/O/W droplets with 100 nM BODIPY dispersed in the oil phase and MreB-RFP in the segmented phase. Fluorescence was detected in separate channels. ]]




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=='''CFS with SWNTs'''==
=='''CFS with SWNTs'''==
==='''AFM'''===
==='''AFM'''===
[[Image:Biomod tianjin results 11.png|center|600px|thumb|Fig 3.AFM analysis of MreB-RFP cell-free aggregation with SWNTs, indicating that SWNTs serve as scaffolds in the spiral winding of fusion proteins in vitro.
[[Image:Biomod tianjin results 11.png|center|600px|thumb|Fig 4.AFM analysis of MreB-RFP cell-free aggregation with SWNTs, indicating that SWNTs serve as scaffolds in the spiral winding of fusion proteins in vitro.
a). AFM image of fusion protein MreB-RFP without SWNT scaffold, protein aggregated in disorder and only discrete spherical particles were formed.
a). AFM image of fusion protein MreB-RFP without SWNT scaffold, protein aggregated in disorder and only discrete spherical particles were formed.
b). AFM image of fusion protein MreB-RFP at the presence of SWNT, protein aggregated
b). AFM image of fusion protein MreB-RFP at the presence of SWNT, protein aggregated
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==='''SEM'''===
==='''SEM'''===
[[Image:Biomod tianjin results 24.png|center|600px|thumb|Fig 4. SEM analysis of MreB-RFP cell-free aggregation with SWNTs.
[[Image:Biomod tianjin results 24.png|center|600px|thumb|Fig 5. SEM analysis of MreB-RFP cell-free aggregation with SWNTs.
a). SEM image of SWNT, the sidewall of nanotube is smooth
a). SEM image of SWNT, the sidewall of nanotube is smooth
without any absorption or aggregation.
without any absorption or aggregation.
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==='''TEM'''===
==='''TEM'''===
[[Image:Biomod tianjin results 3.png|center|600px|thumb|Fig 5. TEM analysis of MreB-RFP cell-free aggregation with SWNTs.
[[Image:Biomod tianjin results 3.png|center|600px|thumb|Fig 6. TEM analysis of MreB-RFP cell-free aggregation with SWNTs.
a). TEM image of SWNT, the sidewall of nanotube is smooth
a). TEM image of SWNT, the sidewall of nanotube is smooth
without any absorption or aggregation.
without any absorption or aggregation.
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==='''Fluorescence micrograph'''===
==='''Fluorescence micrograph'''===
[[Image:Biomod tianjin results 41.png|center|500px|thumb|Fig 6. Fluorescence micrograph of fusion protein MreB combined with SWNT. Red fluorescence can be observed, indicating the activity of fusion protein were reserved. ]]
[[Image:Biomod tianjin results 41.png|center|500px|thumb|Fig 7. Fluorescence micrograph of fusion protein MreB combined with SWNT. Red fluorescence can be observed, indicating the activity of fusion protein were reserved. ]]


=='''Achievement and Prospect'''==
=='''Conclusion'''==
===A “bottom-up” strategy to build up artificial cell surrogates===
*RFP tagged E. coli actin-like cytoskeleton filaments were cell-free synthesized.
*We successfully synthesized the actin-like cytoskeleton filaments in cell-free system and assembling components to create artificial cell surrogates. Bionanotechnology and microfluidics technology are also combined to artificially mimic the synthesis of sub-cellular structural unit which might contribute to recognizing, exploring and even creating life.
*In vitro behaviors of E. coli actin-like cytoskeleton filaments were well characterized via both microfluidics and SWNTs, which proved our previous hypothesis.
===Brand-new system for study on modular organism===
 
*Normally the study of exogenous genes and proteins could be conducted in certain modular organism like E. coli and yeast. However, the research of structure and lethal components in modular organism sometimes comes to a dilemma, as we cannot find a better chassis, and the commonly-used methods such as deletion is not applicable any more. The cell-free system could serve as an optimum chassis which combines transcription and translation processes and avoid the limit of cell support on the expression of modular organism protein as well as some special noxious proteins.
 
=='''Prospect'''==
===“Bottom-up” strategy to build up artificial cell surrogates===
*We successfully synthesized the actin-like cytoskeleton filaments in cell-free system and assembling components to create artificial cell surrogates. Bionanotechnology and microfluidics technology are also combined to artificially mimic the synthesis of subcellular structural unit which might contribute to recognizing, exploring and even creating life.
===Cell free system for study on modular organism===
*Normally the study of exogenous genes and proteins could be conducted in certain modular organism like E. coli and yeast. However, the research of structure and lethal components in modular organism sometimes comes to a dilemma, as we cannot find a better chassis, and the commonly-used methods such as deletion is not applicable any more. Here, cell free synthesis of MreB, originated from E.coli, facilitated the protein characterization.
===Biological compatibility===
===Biological compatibility===
*It has been already discovered that CNTs (Carbon Nanotubes) are poisonous to many kinds of microorganisms. With the aggregation and spirally binding of filamentous cytoskeleton protein MreB on SWNTs (single-walled carbon nanotubes), their surface physical and chemical properties are enormously modified. With further characterization, this kind of SWNTs may become more compatible to microorganisms, even, and serves as comprehensive nanomaterials in more application areas.
*It has been already discovered that CNTs (carbon nanotubes) are poisonous to many kinds of microorganisms. With the aggregation and spirally binding of filamentous cytoskeleton protein MreB on SWNTs (single-walled carbon nanotubes), their surface physical and chemical properties are enormously modified. With further characterization, this kind of SWNTs may become more compatible to microorganisms, even, and serves as comprehensive nanomaterials in more application areas.  
===Biosensor===
===Biosensor===
*In recent years, with the development of immobilized microorganisms technology, the microbial electrode has come up with the advantage of stable quality and relatively low cost. However, there is still no report about the use of CNT in the construction of microbial electrode. In our project, the SWNTs could be covered by protein MreB and connected with biomembrane. Thus there will be a hope to combine SWNTs with living microorganisms, and create of a new kind of coupling way of intracellular enzyme reaction and electrochemical process.
*In recent years, with the development of immobilized microorganisms technology, the microbial electrode has come up with the advantage of stable quality and relatively low cost. However, there is still no report about the use of CNT in the construction of microbial electrode. In our project, the SWNTs could be covered by MreB protein and then connected with biomembrane. Thus, there will be a hope to combine SWNTs with living microorganisms, and to create of a new kind of coupling way of intracellular enzyme reaction and electrochemical process.
 


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Latest revision as of 20:53, 10 November 2011


Home            Project            Results            Video            Team            Acknowledgements



CFS with SWNTs

AFM

Fig 4.AFM analysis of MreB-RFP cell-free aggregation with SWNTs, indicating that SWNTs serve as scaffolds in the spiral winding of fusion proteins in vitro. a). AFM image of fusion protein MreB-RFP without SWNT scaffold, protein aggregated in disorder and only discrete spherical particles were formed. b). AFM image of fusion protein MreB-RFP at the presence of SWNT, protein aggregated and wound spirally onto the surface of nanotube.

SEM

Fig 5. SEM analysis of MreB-RFP cell-free aggregation with SWNTs. a). SEM image of SWNT, the sidewall of nanotube is smooth without any absorption or aggregation. b). SEM image of MreB-RFP without scaffold, protein aggregated in disorder and only discrete spherical particles were formed. c). SEM image of fusion protein at the presence of SWNT, protein aggregated and wound spirally onto the surface of nanotube.

TEM

Fig 6. TEM analysis of MreB-RFP cell-free aggregation with SWNTs. a). TEM image of SWNT, the sidewall of nanotube is smooth without any absorption or aggregation. b). TEM image of MreB-RFP without scaffold, protein aggregated in disorder and only discrete spherical particles were formed. c). TEM image of fusion protein without SWNT scaffold, protein aggregated and wound spirally onto the surface of SWNT.

Fluorescence micrograph

Fig 7. Fluorescence micrograph of fusion protein MreB combined with SWNT. Red fluorescence can be observed, indicating the activity of fusion protein were reserved.

Conclusion

  • RFP tagged E. coli actin-like cytoskeleton filaments were cell-free synthesized.
  • In vitro behaviors of E. coli actin-like cytoskeleton filaments were well characterized via both microfluidics and SWNTs, which proved our previous hypothesis.


Prospect

“Bottom-up” strategy to build up artificial cell surrogates

  • We successfully synthesized the actin-like cytoskeleton filaments in cell-free system and assembling components to create artificial cell surrogates. Bionanotechnology and microfluidics technology are also combined to artificially mimic the synthesis of subcellular structural unit which might contribute to recognizing, exploring and even creating life.

Cell free system for study on modular organism

  • Normally the study of exogenous genes and proteins could be conducted in certain modular organism like E. coli and yeast. However, the research of structure and lethal components in modular organism sometimes comes to a dilemma, as we cannot find a better chassis, and the commonly-used methods such as deletion is not applicable any more. Here, cell free synthesis of MreB, originated from E.coli, facilitated the protein characterization.

Biological compatibility

  • It has been already discovered that CNTs (carbon nanotubes) are poisonous to many kinds of microorganisms. With the aggregation and spirally binding of filamentous cytoskeleton protein MreB on SWNTs (single-walled carbon nanotubes), their surface physical and chemical properties are enormously modified. With further characterization, this kind of SWNTs may become more compatible to microorganisms, even, and serves as comprehensive nanomaterials in more application areas.

Biosensor

  • In recent years, with the development of immobilized microorganisms technology, the microbial electrode has come up with the advantage of stable quality and relatively low cost. However, there is still no report about the use of CNT in the construction of microbial electrode. In our project, the SWNTs could be covered by MreB protein and then connected with biomembrane. Thus, there will be a hope to combine SWNTs with living microorganisms, and to create of a new kind of coupling way of intracellular enzyme reaction and electrochemical process.
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