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Revision as of 20:30, 31 August 2015
Background
Phage-assisted continuous evolution (PACE)
Phage assisted continuous evolution or PACE is a system designed for the continuous, directed evolution of biomolecules. The principle is that Escherichia coli flow through a lagoon in which the bacteriphage M13 are present and viable. Important to note is that the flow rate of the E. coli is adjusted so that it is faster than their doubling time but not of that of the M13. Another important aspect of this setup is the deletion of gene P3 on M13, which is necessary for infection and proliferation. Instead the gene P3 of the M13 is encoded on the E. coli plasmid under the control of an upstream activating sequence (UAS). To undergo further infection cycles, the initial infectious generation of transgenic phage must activate the UAS by binding their activating domain (AD) to the binding domain (BD). This requires favorable protein-protein interactions between an X provided by the M13 and Y provided by the UAS of the E. coli. As a result, and some additional help from a mutagenesis plasmid, M13 evolves this interaction between X and Y in order to stay in the lagoon. A stronger interaction creates a selection advantage and will be favored over a weaker interaction (figure 1).
The bacterial two-hybrid (BACTH) system
Adenylate cyclase is an enzyme from B. pertussis, the agent of whooping cough, and acts as a toxin that binds to calmodulin in eukaryotes elevating the level of cAMP (cyclic adenosine monophosphate) in the organism. This is a large protein with 1,706 amino acids, however its catalytic activity resides in the first 400 amino acids. The catalytic domain is divided further into two sub-domains: catalytic site (25 kDa: residues 1-224) and the calmodulin binding site (18 kDa: residues 225-399). Interestingly, when T18 and T25 domains are expressed separately, no cAMP is produced. But when two interacting proteins of interest, either cystolic or membrane, are fused to T18 and T25, the domains are brought into close proximity and adenylate cyclase activity is restored.
In a BACTH setup, the gene coding for endogenous adenylate cyclase is deleted on an E. coli strain (cya-). The cya- strain is then transformed with plasmids containing the T25 and T18 hybrids. A positive interaction between two proteins of interest, an X and a Y, restores adenylate cyclase activity of T25 and T18. Newly synthesized cAMP will then interact with the catabolite activator protein (CAP). The cAMP/CAP complex binds to promoters and regulates transcription of several genes (figure 2).
This can be combined with the PACE system as the UAS promoter of P3 in E. coli. The expression of P3 would be regulated by the interaction of any genes of interest from the T18/T25 fusion and evolve over time in the lagoon. This is an enhanced version of PACE system because the proteins of interest are not limited to those that only bind DNA. Quantitative assays can directly measure the level of cAMP synthesized and give insight into how the protein interactions are developing over time in the lagoon. This is a high unprecedented throughput assay, creating a very time efficient continuous system untarnished by human intervention steps, unlike that of phage display libraries. BACTH in combination with PACE is a simple and rapid tool, utilizing simple organisms to create strong and complex protein interactions.
- Cassius Dio, 50:5; quoted from Cassius Dio: The Roman History: The Age of Augustus, (trans.) () Scott-Kilvert I.; reprinted in AA100 Assignment Booklet (), Milton Keynes, The Open University, p.18.
who would not groan at hearing that Roman knights and senators grovel before her (Cleopatra) like eunuchs?