To get around the problem of having bacteria in contact with food or medical devices in the applications of Infector Detector and Cell by Date respectively, we aimed to express reporter DNA constructs within a CFS consisting of various E. coli cell extracts (homemade S30, commercial S30 and commercial S30 pT7) in bulk solution. In parallel, we investigated and attempted to optimize conditions for phospholipid vesicle formation. Future work on our project would then involve combining the two, so that protein expression is achieved inside vesicles that have prolonged chassis lifespan.
In vitro Expression in Bulk Solution
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 system most suitable for our two applications.
Preparation of Homemade S30 Cell Extract Protocol
Preparation of Homemade S12 Cell Extract Protocol
Preparation of Homemade S30 Reaction Mixture
Preparation of Commercially Available S30 Reaction Mixture
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) 3 and Vincent Noireaux et al. (2005). 4
The mineral oil method consists of three stages, regardless of composition or equipment:
- Preparation of lipid-oil suspension
- Emulsification of vesicle contents
- Bilayer formation through sedimentation
Cell-Free in Infector Detector
Cell-Free in Cell by Date
- Kim TW, Keum JW, Oh IS, Choi CY, Park CG, and Kim DM. Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system. J Biotechnol 2006 Dec 1; 126(4) 554-61. doi:10.1016/j.jbiotec.2006.05.014 pmid:16797767.
- In vitro protein expression (Promega)
- Sophie Pautot, B. J. Frisken and D.A. Weitz: Engineering asymmetric vesicles. Proceedings of the National Academy of Sciences 100, p. 10718-10721 (2003).
- Noireaux V, Bar-Ziv R, Godefroy J, Salman H, and Libchaber A. Toward an artificial cell based on gene expression in vesicles. Phys Biol 2005 Sep 15; 2(3) P1-8. doi:10.1088/1478-3975/2/3/P01 pmid:16224117.