Difference between revisions of "Part:BBa K3611009"

Revision as of 01:23, 22 October 2020

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).

https://2020.igem.org/File: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).

https://2020.igem.org/File: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.

https://2020.igem.org/File: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

@@ Line 3: / Line 3: @@
-When there are few input signal, the report signal output by the engineered bacteria will be very weak. In order to ensure people’s health and safety as much as possible, we hope the detection limit of our detection system is low enough and the output level is high enough to ensure that trace viruses would not be missed. In order to achieve this, we designed a cascade amplifier to amplify the detected virus signal, to increase the expression of the reporter gene and lower the detection limit.
+'''Usage and Biology'''
-The cascade amplifier comprises two modular terminals—the input and the output. It consists of two independent orthogonal amplifiers arranged in series, which are constructed using hrp operon from Pseudomonas syringae, and extracytoplasmic function (ECF) σ factor ECF11_987 and promoter ECF11_3726 from Vibrio parahaemolyticus.
-The natural hrp regulatory network contains activator protein HrpR and HrpS. These two would form an ultrasensitive high-order co-complex HrpRS, which bind the upstream activator sequence of the hrpL promoter to remodel the closed σ54-RNAP-hrpL transcription complex to an open one through ATP hrdrolysis. This have been proven to amplify the input transcriptional signal.
-Bacterial sigma factors (σs), the promoter recognition subunits of RNA polymerase (RNAP), are modular proteins with domains that recognize DNA sequences in the -10 and -35 regions of their target promoters. In addition to the housekeeping σs that recognize the thousands of promoters, bacteria have a variable number of stress activated alternative σs that direct RNAP to distinct promoter sequences. Extracytoplasmic function (ECF) σs are one type of them. By comparison promoters of ECFs are highly conserved, which means they could only be specificity activated by their own corresponding ECFs. The promoters also have a large dynamic range of output, where the OFF state is very low in the absence of the σs and the ON state produces a high level of expression, which could be used as a signal amplify part. We select ECF11_987 σs and promoter ECF11_3726, which are proved as a higher-gain amplifier combination, to build our secondary amplifier.
-In principle, the transduced transcriptional signal of the sensor could be sequentially amplified through multilayer coupled amplifiers. We connect two amplifiers in series. The first amplifier’s output is the second amplifier’s input, so that the second amplifier could amplify the first one’s output signal. In general, the initial sensor’s signal will be amplified twice.
+'''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).
+https://2020.igem.org/File: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).
+https://2020.igem.org/File: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.
+https://2020.igem.org/File: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.