Abstract

Reguli

Biological engineering has brought us handy tools to produce desired products. However, we have to induce genes to properly express them on correct cell phase. We, KAIST 2012 iGEM team, suggest a module that automatically turns the transcription of gene on or off using dual-phase switching module. In our model, promoter orientation is designed to be reversed when sufficient quorum molecule is detected. Then, genes on the other side, indigo synthesizing enzyme bFMO, lactonase, and excisionase are expressed. Indigo is meaningful in that it is the end-product of its metabolic pathway and, its color can be easily detected. Lactonase quenches quorum molecule(lactone), which will generate an oscillating pattern. Excisionase acts on the recombination site to set promoter to its initial orientation. In this way, we can regulate metabolic pathways. So we call it regulative E.coli, Reguli.

Introduction

"We suggest an auto-regulation module free from induction which utilizes dual-phase switching system."

Throughout past iGEM competitions, many kinds of bio-modules were proposed and tested. They were great and some were brilliant, but it doesn’t seem many of them are universally available. Of course the goal of iGEM is to enrich the database, but we view the beauty of this registry is to contain as many modules which can be readily applied to actual research as possible. Thus, here now, we suggest an auto-regulation module free from induction which utilizes dual-phase switching system and quorum sensing.

We may think of many meanings for this module; bio-computing, in-cell signal processing, and auto-control of cell metabolism. For example, since we use dual-phase switching system, we may consider each direction of gene expression as a signal; signal 0 or 1 of binary code. Thereby we can use this module to generate in vivo logic gates or computational system.

Dual-phase switching system adopts DNA recombination system of bacteriophage origin. In this system, DNA integrase recognizes specific sequences called attB and attP and then invert the sequence between them. Exisionase revert this sequence into its original state by recognizing the recombination site. We can thereby use this system to turn the pathway on or off.

Products of LuxI and LuxR are used to generate signals in our quorum sensing model. They initiate the operation of auto-regulation module. Lactonase will regulate the signal to yield an oscillating pattern.

As our end product, we selected bio-indigo which comes from indole. This widespread pigment is produced by bFMO, bacterial Flavin-containing MonoOxygenase. Successful production of bio-indigo verifies that our module can be embodied well in metabolic pathway and also proposes its potential usage.