Biomod/2011/Tianjin:Results: Difference between revisions

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=Results=
==''' Cell-free synthesis of actin-like cytoskeleton filaments'''==
=='''CFS with microfluidics'''==
===''' 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.
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. ]]
[[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'''===
=='''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.
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. ]]
[[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. ]]

Revision as of 04:29, 1 November 2011


Home            Project            Results            Video            Team            Acknowledgements

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.

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.

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.


CFS with SWNTs

AFM

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. 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 4. 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 5. 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 6. Fluorescence micrograph of fusion protein MreB combined with SWNT. Red fluorescence can be observed, indicating the activity of fusion protein were reserved.


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