Composite

Part:BBa_K3611009

Designed by: Peixuan Zheng   Group: iGEM20_NEU_CHINA   (2020-10-20)
Revision as of 01:42, 22 October 2020 by Arcdia (Talk | contribs)

A cascade transduction signal amplifier


Usage and Biology

Cascade Amplifier

This year, 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. 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 (Fig. 1).

T--NEU_CHINA--Cascade_amplifier_in_detail.png

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).

T--NEU_CHINA--mp137.png

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.

T--NEU_CHINA--Cascade_Amplifier_2.png

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.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1897
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1879
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1089
    Illegal SapI.rc site found at 1722


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