In lab, we prepared synthetic transcription-translation systems to synthesize GFP or FlAsH peptides.
There was high background fluorescence (although I'm not sure what wavelength the background was measured for).
designing a synthetic minimal cell
Synthetic minimal cells (SMCs) or in vitro transcription/translation (TXTL) systems can be beneficial for many purposes, such as for the production of large amounts of proteins, or production of proteins that might be otherwise harmful to a cell. I would want to design a minimal cell to produce antimicrobial peptides, or AMPs. AMPs are peptides that can be produced by plants, animals, and microbes, and target and kill bacteria by either disrupting the membrane or inhibiting intracellular functions. AMPs can be acquired by isolating them from the organism in which they're produced (so the amount of peptide you collect would depend on their production by the organism); by ordering synthetic peptides; or by introducing the coding sequence to a bacterial expression vector (as long as the AMP is not harmful to the bacteria).
Previously, Martemyanov et al. have used a cell-free system to produce the antimicrobial peptide Cecropin, but the cecropin had to be extracted after production. I would like to try producing a protease-activated antimicrobial peptide (PAMP), which is secreted after being produced by Proponibacterium jensenii and requires proteolytic activation by external proteases.
Function 1A. The synthetic cell would produce PAMP and the sec-dependent pathway to export it. 1B. This could possibly be done with cell-free TX/TL without encapsulation, which would eliminate the need for the secretion system. However, since PAMP is activated by protease activity, it might be that if the TX/TL system was not encapsulated, the protease might act on the PAMP before its translation was completed, which might affect the activity. 1C. This could be done in a genetically modified natural cell, but the sec pathway is large, around 14 genes, so introducing that many genes to a cell might be difficult. 1D. The desired outcome is that functional PAMP peptides are produced and secreted, so if the synthetic cell is mixed with targets of PAMPS (Propionibacteria and Lactobacilli) it should cause killing of those bacterial cells.
Components 2A. Membrane: phospholipids, cholesterol (typical membrane bilayer). 2B. Encapsulate: cell free TX/TL system, genes for PAMP and sec pathway.
2C. Organism TX/TL system should come from: bacterial, since PAMPs are produced in bacteria. 2D. Cell interact with environment: would need to express sec pathway, which involves a membrane channel and targeting signal.
Experimental details 3A.
Lipids: cholesterol
Enzymes: bacterial TX/TL system
Genes: PAMP pamA gene (Faye et al., 2002) and sec pathway (Pugsley, 1993) 3A.Test for function with a bacteria killing assay.
As a side note, I wonder if SMCs can be used to introduce mutations to DNA (eg by exposing them to UV), and introduce mutations to genes of known antimicrobial peptides to see if more potent ones are produced? Also, not certain that the sec pathway would be functional in a non-bacterial cell?
