User:Cheuk Ka Tong/Sandbox5

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Design

To get around the problem of having bacteria in contact with food or medical devices in the applications of Cell by Date and Infector Detector respectively, we aimed to express reporter DNA constructs within a CFS consisting of cell extract encapsulated in selectively permeable vesicles. Our plan was to first prepare the cell extract and perform in-vitro expression in bulk solution. In parallel, we would investigate and optimize conditions for phospholipid vesicle formation. The final stage of our project would involve combining the two, so that protein expression is achieved inside the vesicles.

In-Vitro Expression

Since we are using E. coli plasmid DNA constructs, we chose to use the compatible E. coli cell extract for in-vitro expression. We tried out four options so as to find out the chassis most suitable for our two applications.

Homemade
Commercial
E. coli S30 extract
E. coli S12 extract
E. coli S30 extract
E. coli T7 S30 extract
  • Prepared using the protocols described by Kim DM et al. (2006)
  • Used E. coli strain BL21 (DE3) with loss of function mutations in OmpT endoproteinase and lon protease genes
  • BL21 can express T7 RNA polymerase under the control of LacI, inducible by IPTG
  • Protocols for preparation of E. coli S30 and S12 extracts
Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system by Kim DM et al. (2006)
Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system by Kim DM et al. (2006)
  • Purchased from Promega
  • Prepared by modifications of the method described by Zubay et al. (1980)
  • Used E. coli strain B deficient in OmpT endoproteinase and lon protease activity







Vesicle Formation

For the formation of phospholipid bilayer vesicles, we chose to use the mineral oil method. The protocols are based on Engineering Asymmetric Vesicles by Sophie Pautot, BJ Frisken, and DA Weitz. (2003) and Toward an artificial cell based on gene expression in vesicles by Vincent Noireaux et al. (2005).


Toward an artificial cell based on gene expression in vesicles by Vincent Noireaux et al. (2005)
Toward an artificial cell based on gene expression in vesicles by Vincent Noireaux et al. (2005)

Summary of the mineral oil method
The mineral oil method consists of three stages, regardless of composition or equipment:

  1. Preparation of lipid-oil suspension
  2. Emulsification of vesicle contents
  3. Bilayer formation through sedimentation

Detailed protocol for vesicle formation


In-veso chassis development

Below is a simplified timeline of the in-veso chassis development process and its integration with the rest of the project.

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