Endy:Chassis engineering/Orthogonal protein synthesis

Dedicated systems

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

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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

Reporter devices for the transcription/translation systems

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

Current Work

1. Demonstrate specificity of combined dedicated systems - Combinations of dedicated systems should allow highly specific production of proteins with no non-specific protein production by E. coli systems. This can be demonstrated by stably maintaining a plasmid-borne copy of a highly toxic gene in a chassis. It should be possible to turn on high level expression of this toxic gene by inducing the dedicated systems.
   • The current candidates for a toxic gene are CCDB, T7 gene 5.3 or T7 gene 7.7.
   • If you have a gene you would like to stably maintain with zero expression, let me know and I'll have a go!
   • A quick check to see if this will work can be done by looking at the expression level of GFP when the dedicated systems are not induced. The initial attempt to show this didn't look good, expression level of GFP from E7103 was the same as E7104 and both were a little bit higher than BL21(VM1.0) with no GFP coding plasmid. This may be due to the fact that the expression of the dedicated ribosomes required by E7103 is leaky due to the P_BAD promoter. To get around this, I'm going to redo this experiment using glucose as a carbon source to further repress the promoter.
2. Demonstrate decoupled function of the system and the cellular chassis - While the above point shows that the dedicated systems are highly specific, the real objective of dedicated systems is to decouple the function of system and cellular chassis, such that perturbations to one should not be transmitted to the other. For example a sudden increase in the protein production rate of the system should not cause the chassis to stop growing. Alternatively a reduction in the growth rate of the chassis due to a scarcity of nutrients or another environmental effect should not affect the rate of protein accumulation in the system.
   • 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.
3. Better understanding of dedicated system performance - I'm still trying to understand some of the details of how the systems are operating.
   • The initial rate of GFP accumulation seems to be same when using dedicated translation and chassis translation. Since there was no attempt to make sure that there were the same numbers of ribosomes available for each or to make the RBS strenghts the same it is unlikely that the translation rates for reporters using both should be the same. The most likely explanation appears to be that some factor downstream of translation machinery is limiting reporter production. Here are some possibilities -
     • Supply of charged tRNAs. This would assume that there are large numbers of transcripts and large numbers of ribosomes translating the messages such that the production is being limited by the supply of charged tRNAs.
     • Alternatively, translation may not be limiting at all but some step related to folding of the GFP peptide is limiting. Not sure what chaperones are involved in GFP folding and maturation.