Biomod/2011/Tianjin:Project: Difference between revisions
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*In a cell-free system, coupled transcription-translation systems usually combine a bacteriophage RNA polymerase and promoter with eukaryotic or prokaryotic extracts rich in ribosomes, transfer RNAs and aminoacyl tRNA transferase enzymes. Buffers are also added to maintain the appropriate magnesium and salt concentrations required for efficient translation. Protease inhibitors can be added to minimize degradation of synthesized proteins. In addition, an ATP regenerating system involving is used to power and prolong the lifespan of the expression machinery. Simply by adding the DNA template to the cell extract and feeding solution, the CFS would be able to express the encoded genetic circuit. | *In a cell-free system, coupled transcription-translation systems usually combine a bacteriophage RNA polymerase and promoter with eukaryotic or prokaryotic extracts rich in ribosomes, transfer RNAs and aminoacyl tRNA transferase enzymes. Buffers are also added to maintain the appropriate magnesium and salt concentrations required for efficient translation. Protease inhibitors can be added to minimize degradation of synthesized proteins. In addition, an ATP regenerating system involving is used to power and prolong the lifespan of the expression machinery. Simply by adding the DNA template to the cell extract and feeding solution, the CFS would be able to express the encoded genetic circuit. | ||
*Despite the lack of experience and characterization, CFS shows huge advantages on the expression, control and purification special exogenous proteins which interrupt the normal metabolism of host cell. Here CFS is particularly suited for constructing artificial cell surrogates. | *Despite the lack of experience and characterization, CFS shows huge advantages on the expression, control and purification special exogenous proteins which interrupt the normal metabolism of host cell. Here CFS is particularly suited for constructing artificial cell surrogates. | ||
===CFS and | ===CFS and Microfludics=== | ||
*To mimic the natural cell more faithfully, cell-free expression systems have been combined and encapsulated into double emulsion droplets, which is produced by microfluidcs technology. Water in oil in water (W/O/W) droplets are created by using a hybrid Lab-chip devices comprising a hydrophobic network (supporting a continuous oil phase), interfaced with a hydrophilic network (supporting an aqueous phase). | *To mimic the natural cell more faithfully, cell-free expression systems have been combined and encapsulated into double emulsion droplets, which is produced by microfluidcs technology. Water in oil in water (W/O/W) droplets are created by using a hybrid Lab-chip devices comprising a hydrophobic network (supporting a continuous oil phase), interfaced with a hydrophilic network (supporting an aqueous phase). | ||
[[Image:Biomod tianjin pro 3.png|center|400px|thumb|Fig 3.Double emulsion generation: a microfluidic system made of two glass chips with different wetability characteristics.]] | [[Image:Biomod tianjin pro 3.png|center|400px|thumb|Fig 3.Double emulsion generation: a microfluidic system made of two glass chips with different wetability characteristics.]] | ||
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====Cell-free expression in W/O/W microdroplets==== | ====Cell-free expression in W/O/W microdroplets==== | ||
*After the expression of gene cassette, we proceeded with the combination of cell-free system with the microfluidics technology to see whether the synthesis of MreB in vitro could continue in microdroplets and assign to the oil phase. Two different DNA templates were used, fusion protein MreB-RFP to study the protein’s phase separation at the membrane, and the water soluble RFP as a control. | *After the expression of gene cassette, we proceeded with the combination of cell-free system with the microfluidics technology to see whether the synthesis of MreB in vitro could continue in microdroplets and assign to the oil phase. Two different DNA templates were used, fusion protein MreB-RFP to study the protein’s phase separation at the membrane, and the water soluble RFP as a control. | ||
==== | ====MreB aggregation with SWNTs==== | ||
*The fusion protein MreB-RFP would be affiliated to SWNTs after surface treatment and its aggregation patterns is mainly determined by the nickel ions distribution on the sidewall and the polymerization state of itself. | *The fusion protein MreB-RFP would be affiliated to SWNTs after surface treatment and its aggregation patterns is mainly determined by the nickel ions distribution on the sidewall and the polymerization state of itself. | ||