Endy:Chassis engineering/Orthogonal protein synthesis

Dedicated systems

Idealized dedicated systems make the protein production processes for the engineered system and the cellular chassis' own systems entirely orthogonal

• Dedicated systems are a method to decouple the function of an engineered biological system from the function of its chassis.
• By separating the resources and machinery used to supply and power an engineered system from those of the chassis, then perturbations in the operation of one should have less effect on the other.
• Currently, we are working to implement these systems at a transcription and translation level.
• These dedicated synthesis systems can then be used as the basis of Biological virtual machines
• The different methods we are using to implement dedicated systems are described in more detail below. We have built reporter devices to examine the performance of the four different combinations of transcription systems currently available to us. They are shown in matrix form below.

Reporter devices for the transcription/translation systems

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

VM Transcription

Introduction

The T7 expression system developed by Studier and coworkers is essentially orthogonal from the E. coli transcription system. T7 RNAP does not recognize E. coli promoters and E. coli RNAP does not recognize T7 promoters.

Effects of producing dedicated transcription machinery in a cellular chassis

Growth curves for three colonies of BL21(DE3) in the presence or abscence of maximally inducing levels of IPTG.

Three colonies of BL21(DE3) were grown in the presence or absence of 0.4mM IPTG. The IPTG induces the production of T7 RNAP. Results suggest that the presence of dedicated transcription machinery in BL21 does not noticeably affect the growth rate of the cellular chassis.

While this implies that the presence of a dedicated transcription system has negligible effect on a cells ability to supply a demand, it is possible that the added demand of these systems pushs the cellular chassis much closer to its maximum demand level before physiological changes become evident.

Dedicated Translation

RBS design

The first RBS I designed was BBa_B0036. This part uses the RBS described by Brink et al.. The spacing between the RBS and the start codon was set to be the same as that of the consensus E. coli RBS sequence.

The RiPS from BBa_B0036 are not as high as I would like. Hence I am designing a new RBS. For this new RBS, I will use the spacing between RBS and start codon used by Brink and coworkers. I have obtained high levels of translation using this spacing as shown in the protein gel below using the reporter construct pSD-rbsK2 (obtained from Chris Hayes)

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.