Team:HUST-China/Description

Application

Description

OVERVIEW

This year, we want to offer a handy, adjustable and useful toolkit to everyone working on Synthetic Biology--Signal Filter. The core circuit is based on a positive feedback bi-stable or tri-stable system with the capacity of reducing noise and converting pulse signal into robust and persistent signal. To adapt to different experimental requirements, we also developed two versions of our Signal Filter--prokaryotic one and eukaryotic one.

Prokaryotic version:

It is a tri-stable system adapted from bacteriophage λ operon. In the operon, promoter RE is activated by the transcriptional activator CII. Ftsh is an ATP-dependent host metalloprotease which will normally degrade CII, while CIII serves as an inhibitor of Ftsh to free CII. And CI can function as an inhibitor to block pR, while Cro can bind pRE to stop the downstream gene’s expression. The positive feedback control under pRE is used to enhance pulse signal 1 and convert it into a robust and stable signal.

Fig*: Circuit of prokaryotic version serving as a tri-stable signal filter

When signal 1 comes, pRE will be activated, expressing Cro and CII. At the same time, the constitutively produced CIII will guarantee enough CII to enhance the transcription downstream the pRE. Thus,there forms a positive feedback loop to direct a fast and strong expression of Cro. A quantity of Cro can repress the transcription under pRM by binding to Cro binding site and blocks gene of interest 2’s expression, so it turns to a stable state of expressing gene of interest 1.

When signal 2 comes, under a certain inducible promoter, CI will be expressed and then binds to CI binding site, blocking the expression of gene of interest 1 and cIII. With CIII’s reduction, FtsH gradually degrades CII and interrupts the stable state. Therefore, Cro’s expression will immediately drop down, allowing the expression of gene of interest 2. So the system turns to another stable state.

If there is no input signal, both genes of interest will be expressed. And if both of the two signals exist, the expression state will depend on the intensity of the initial input signal.

Eukaryotic version:

It is a bi-stable system derived from Arabidopsis thaliana stress response system. Enzyme catalysis is the core of this design to increase the efficiency of state transition.

Fig*: Circuit of Eukaryotic version serving as a bi-stable signal filter

Clade A protein phosphatases type 2C(PP2CA) and SUCROSE NONFERMENTING1-RELATED SUBFAMILY2(SnRK2s) protein kinase are both components of Abscisic acid signaling network in Arabidopsis thaliana. ABF2 is a leucine zipper transcription factor which basically binds to ABA-response element (ABRE). ABF2 can be phosphorylated by SnRK2s and efficiently dephosphorylated by PP2CA. Also, in the absence of Abscisic acid, SnRK2 kinases will be inactivated by PP2Cs and thus, efficiently shut down the system.

When signal 1(ON) comes, promoter RD29A drives the expression of SnRK2.2, which can phosphorylate ABF2 (constitutively express). Comparing to protein co-facter association, enzyme catalysis can produce a large quantity of phosphorylated ABF2 in a short time, then enhances pRD29A to turn on the gene of interest’s expression. When signal 2(OFF) comes, gene PP2CA expresses, dephosphorylating ABF2 and inactivating SnRK2.2, thus turning the system into OFF state.