Endy:Chassis engineering/Orthogonal protein synthesis

Motivation

An independent protein production channel, dedicated to the synthesis of engineered system proteins would contribute to decoupling the operation of systems and their cellular chassis

Engineered biological systems use host cells as a power supply and chassis. In an ideal chassis-system relationship, the operation of the chassis should be decoupled from the operation of the system and vice versa. This is not the case for today's engineered biological systems. For example, today's engineered systems use the same transcription and translation machinery as the cellular chassis. The engineered system and the chassis also share a pool of nucleotides, amino acids, and tRNAs. Variations in machinery and material levels affect the operation of the engineered system and high demands for machinery and materials by the system perturb the function of the chassis.

A solution to this problem of the coupled function of the engineered system and chassis is to construct a second protein production system inside the chassis, which is independent from, or orthogonal to, the endogenous protein production system. Ideally, this dedicated system channel would use transcription, translation machinery that is orthogonal from that of the cellular chassis and also draw from a different material pool than the chassis. The levels and regulation of the components of the dedicated system channel could be modified independent from the levels and regulation of the protein production system of the chassis itself.

If this dedicated system channel could be standardized and designed to be able function in multiple cell types, then the dedicated systems could form the basis of a biological virtual machine

Currently, we are working to enable dedicated transcription and dedicated translation in an E. coli strain MG1655 chassis. Details on this work can be found on the following pages -

• Dedicated Transcription
• Dedicated Translation

Demonstrating the usefulness of dedicated systems

1. Measure specificity of combined dedicated systems
2. Demonstrate decoupled function of the system and the cellular chassis
   • GFP accumulation as chassis make the transition from log to stationary phase should indicate whether system performance is less affected by the change in the chassis state.
   • Suddenly turning on the high level protein expression of a system should reduce growth rate of the chassis when the system uses dedicated systems than when it does not. Initial experiments have suggested that this might only be true in a minimal media such as M9.

First generation reporter devices for the dedicated transcription/translation systems

	General Translation	VM Translation
General Transcription	I7101	I7102
VM Transcription	E7104	E7103