Biomod/2011/Tianjin:Results
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Contents
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.
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.
CFS with SWNTs
AFM
SEM
TEM
Fluorescence micrograph
LCSM
Achievement and 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.