Difference between revisions of "Part:BBa K3611009"

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A cascade transduction signal amplifier

Usage and Biology

Cascade Amplifier

We connected a secondary amplifier in series on the basis of the original hrp amplifier. We select extracytoplasmic functional (ECF) sigma factor ECF11_987 and promoter ECF11_3726, which are proved as a higher-gain amplifier combination, to build our secondary amplifier[1].

Two independent quadrature amplifiers connected in series form a cascade amplifier. The output of the first amplifier is the input of the secondary amplifier, so the second amplifier can amplify the output signal of the first amplifier. Generally, the signal of the initial sensor will be amplified twice [2] (Fig. 1).

Fig. 1 The cascade amplifier consist of hrp amplifier and ECF-based amplifier. LacUV5 promoter, the gene downstream of this promoter will be transcribed under the IPTG activation. The hrpR and hrpS are activator protein’s coding genes, PhrpL, a promoter which can be induced by the ultrasensitive high-order co-complex HrpRS. ECF is the coding gene of ECFs ECF11_987, while the Pe11, promoter ECF11_3726 can be activate by it. The ribosome binding sites (RBS) B0030 and terminator B0015 were indicated as gray. The enhanced fluorescent protein (EGFP) was used as our reporter protein, with egfp as its coding gene.

Compared with a separate hrp gene regulatory network, the cascade amplifier can better amplify the detected signal, increase the expression of the reporter gene, and reduce the detection limit (Fig. 2).

Fig. 2 The cascade amplifier and its characterization graph. Cells are induced by 5 varying concentrates of IPTG (0, 10-6, 10-5, 10-4, 10-3 mmol/L) after 1h

After-Translational Regulatory

Due to our cascade amplifier, not only the inducible expression level was raised, but also the basal expression, which also called leakage. To reduce the amplifier’s raising-leakage effect, we integrated a protease-based post-translational degradation control system. First a protein degradation tag (AAV) is added to the reporter protein to reduce the output basal expression. To obtain low basal level without sacrificing the high output, we next incorporated the sensor into a TEV protease-based reporter protein degradation control system (Fig. 3). The TEVp will be activated to be expressed by the same transduction input signal as cascade amplifier, which can cleave the linker between the expressed reporter protein and its fused AAV degradation tag [3].

Fig. 3 The cascade amplifier with TEVp-based protein degradation system. The PmrC promoter is the output of ACE2-PmrCAB detection system. With the low-background cascade amplifier, the output would maintain a high output and low leakage.

Pitifully, we don’t have enough time to construct this after-translation regulatory system. But we modeled the part in the dry lab work and demonstrated that it can work very well. Please click here to gain more details.

Reference

[1] Rhodius, V. A. et al. Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters. Mol. Syst. Biol. 9, 702 (2013).

[2] Xinyi Wan,Francesca Volpetti,Ekaterina Petrova,Chris French,Sebastian J. Maerkl,Baojun Wang. Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals[J]. Nature Chemical Biology,2019,15(5).

[3] Fernandez-Rodriguez, J. & Voigt, C. A. Post-translational control of genetic circuits using Potyvirus proteases. Nucleic Acids Res. 44, 6493–6502 (2016).

Sequence and Features