Targeted proteomics measurements for Genetically controlled membrane synthesis in liposomes
Blanken D, Foschepoth D, Serrão AC, Danelon C. Genetically controlled membrane synthesis in liposomes. Nat Commun. 2020 Aug 28;11(1):4317. doi: 10.1038/s41467-020-17863-5. PMID: 32859896; PMCID: PMC7455746.
- Organism: Escherichia coli
- Instrument: 6460 Triple Quadrupole LC/MS
PURE, cell free, synthetic cell, gene expression, lipid biosynthesis
Lab head: David Foschepoth
Submitter: David Foschepoth
Lipid membranes, nucleic acids, proteins, and metabolism are essential for modern cellular life. Synthetic systems emulating the fundamental properties of living cells must therefore be built upon these functional elements. In this work, phospholipid-producing enzymes encoded in a synthetic minigenome are cell-free expressed within liposome compartments. The de novo synthesized metabolic pathway converts precursors into a variety of lipids, including the constituents of the parental liposome. Balanced production of phosphatidylethanolamine and phosphatidylglycerol is realized, owing to transcriptional regulation of the activity of specific genes combined with a metabolic feedback mechanism. Fluorescence-based methods are developed to image the synthesis and membrane incorporation of phosphatidylserine at the single liposome level. Our results provide experimental evidence for DNA-programmed membrane synthesis in a minimal cell model. Strategies are discussed to alleviate current limitations toward more effective liposome growth and self-reproduction.
In vitro protein expression reactions using the PURE system were conducted with multi-gene plasmids containing the genes for E.coli lipid synthesis under control of two phage promotors for orthogonal expression. The proteomics data was used to verify the orthogonality and to measure a response to RNA polymerase concentrations
THe sample is consisting of PURE cell free expression system obtained from GeneFrontier (japan). Purified DNA with T7 or SP6 promotor can be readily expressed in the system and in solution digestion with trypsin can be conducted without a a problem.
Created on 7/16/20, 5:08 AM