Overview

Why does Autoregulation so necessary?

When we bring external pathway into E.coli to mass-produce the protein, it may collide with E.coli’s innate pathways by consuming critical resources which generate the redox potential (ATP, NADH, or NADPH molecules). This means that E.coli has to reach certain reducing potential to perform exotic metabolic pathway in the cell.

In conventional methods, the promoters controlling protein expression are repressed until the cells get capacity to produce the protein. Then, this promoter can be induced by putting inducer to the solution on proper cell phase; cell phase can be measured by O.D. value. This kind of method was used because we assume that cell cultural Optical Density (which synchronizes with cell number in the culture) represents inner-cell reduction potential. Although in many time this method successfully makes cells to induce, there are several downsides in this method.

First of all, problem arises with the process of adding inducers like IPTG or arabinose into media. Because the induction time is decided only by considering cell density (O.D.value) not the capacity of the cells (reduction potential), products may not come out as we predicted. Also, it is an annoying job to measure optical density, finding optimal induction conditions. Even more, if we miss the right time point, we might have to throw them away and start from the first place...

This is why we came up with our auto-regulative device. As the result of iGEM project this summer, we suggest an auto-regulation module free from induction which utilizes dual-phase switching system.

Using the FlipFlop device, we can generate biological machine that controls the metabolism as we intended. We decided to call the system as "Auto-regulating system in E.coli", Reguli. To demonstrate the idea, we applied the device to control bio-indigo production, which is catalyzed by bacterial flavin-containing monooxygenase(bFMO).

What is bFMO?

Bacterial flavin-containing monooxygenase(bFMO) converts indole into isatin, which is then sequentially catalyzed into indigoid compounds emitting indigo color.
Because the enzyme utilizes the primary metabolite, we can easily notice the enzyme is working well or not. For the following experiments, engineered bFMO gene from Methylophaga sp. Strain SK1 is kindly provided by Duhee Bang from Yonsei University, Republic of Korea.

Working scheme of Our Design