Application for Speaker (SEED2015)

Evolving around the genetic switches and circuits.

Daisuke Umeno

Graduate School of Engineering, Chiba University, Japan,

E-mail address: umeno@faculty.chiba-u.jp

PRESTO, Japan Science and Technology Agency (JST), Japan

Abstract

Directed evolution is a powerful strategy to construct complex yet functional systems ranging from genetic switch, regulatory circuits, and metabolic networks. In the last SEED meeting, we developed and tested various types of positive/ negative selection systems to widely meet the demand for improving switching stringency, PoPS values, and rapidity in on/off transition. In this meeting, we would like to show various applications of our systems ranging from new induction systems to rapidly tuning their device specificities, as well as their gathering into multi-input genetic circuits.

1. Evolving Switches.

To create new induction system that strictly meet the demand from industrial sides â?? self cloning, use of safe and cheap induction reagents, high induction level, and high stringency-, we created plasmid-encoded regulatory systems based on Bet-operon. Due to the need to minimize the interactions with chromosome-encoded Bet operons, the prototype Bet-switches were implemented on high-copy vectors and under highly active promoters, resulting in the â??always-offâ?? behaviour. Random mutagenesis of BetI genes, followed by ON-/ OFF- selection in the presence/ absence of choline, resulted in the isolation of dozens of switches with different properties (PoPS, stringency, IC50, â?¦.etc). Multiple round of directed evolution also (and quickly) isolated various types of Bet- switches with (1) altered ligand specificity and/or (2) reversed phenotypes (where choline acts as co- repressor). In this presentation, we also show the directed evolution of various known switches to
â??betterâ?? behaveâ?? in various context.

2. Two-input gates and circuits.

Even the complex and multi-membered circuits ultimately turn-on and -off the gene expression. The switches we created was assembled into the prototype logic gates and subjected to the
randomization and systematic on/ off selection in various input states. Almost all of the starter
circuits malfunctioned, but we obtained rapidly obtained a series of two-input gates (AND, imply, N-imply, NAND, â?¦.etc) with decent fidelity/ robustness.