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.

Auto-regulative device


With our automatically-regulative device above, we don’t have to do anything at all to induce gene. When the cells have the capacity to produce the target molecule, the pathway will be turn on. When the cell lost the capacity it will turn off. This is done by cell-source sensor and sensor-generated integrase gene. Integrase and excisionase invert and retrieve the recombination site which exists on the left and right side of promoter. After integrase gene inverts the promoter orientation, genes encoding bio-product pathway are transcribed, making bio-products. In addition, excisionase gene is expressed, reversing promoter orientation to its original state.

Environmental Impact


Auto-regulating device can be further applied to make a noble sensing/degrading device for toxic compound or pollutant. When cells detect certain amount of toxic or pollutant, sensor induce integrase gene, thus invert the promoter sequence in between two recombination sites. After that, scavenger is generated degrading toxic/pollutant molecule. This novel sensing/scavenging device has its advantage in taking care of the environment and relieving the stress of host cell in automatic manner.

1. Promoter : BBa_J23119
2. RBS : BBa_B0034
3. att sites : Recognition site for BBa_K907000(Mycobacteriophage Bxb1 integrase).

Initially, the FlipFlop promotes the transcription and translation of down-stream gene Protein A according to its promoter orientation. When BBa_K907000(Mycobacteriophage Bxb1 integrase, Bxb1_Int) recognizes and inverts the sequence flanked by attB and attP sequences, promoter orientation is reversed, leaving recombined attL and attR sequences.

Then this device promotes the transcription and translation of upstream gene(must be designed in reverse orientation at construction step), or Protein B(Signal 1) in Figure 4.


Also, the signal can be restored to its initial state when BBa_K907000/ BBa_K907001 (Mycobacterio-phage Bxb1 excisionase, Bxb1_Xis) complex recognizes and inverts the sequence flanked with attL and attR sequences back to original state.

Reguli

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

At its initial state (while signal = 0), downstream gene of promoter, LuxI, produces AHL(N-Acyl homoserine lactone). Separated from FlipFlop device, LuxR gene is transcribed under the control of constitutive promoter. Generated LuxR molecules and AHLs form complex to initiate the transcription of Bxb1 integrase, positioned at the downstream of pLuxR promoter. Then, Bxb1 integrases specifically bind to attB and attP sequences to generate signal 1.

At its inverted state(while signal = 1), bFMO gene of upstream sequence can be transcribed and translated. So that bio-indigo starts to be produced. Upper genes of bFMO, AHL-Lactonase and Bxb1 Excisionase are expressed in order, degrading AHL-LuxR complexes and forming Bxb1 integrase-excisionase complexes, respectively. Finally, Int-Xis complexes bind to attL and attR sequences and invert the promoter into its original orientation, regenerating attB and attP sequences.