Difference between revisions of "Part:BBa K2967012"

Latest revision as of 15:13, 27 October 2020

The fixed-gain bio-amplifier

The tunable biological amplifier (Fig. 1) comprises three modular terminals--the input, the output and a gain-tuning input. The device can continuously process the input transcriptional signal with an externally tunable gain (the amplification ratio of the changes in output to input) control.[1]

Figure 1. The composition of tunable biological amplifier. Transcription input can be amplified through the amplifier.

At first, we construct the fixed-gain amplifier (Fig. 2) for the prework. In order to reduce the impact of exotic microorganisms' colonizations in the intestine and to accelerate the secretion of IL-10 and myrosinase, we found a gain-tunable transcription amplifier in Pseudomonas syringae to tune amplifier the input signal.

To verify the fixed-gain amplification(Fig. 2) capability, we integrated the T7 promoter as the input of the fixed-gain amplifier with GFP as the output. When the transduced transcriptional input from the T7 promoter was connected to our amplifier, the resulting output signal amplitude and dynamic range increased significantly as well as the response sensitivity to the inducer (Fig 3.).

Figure 2. Diagram for fixed-gain amplifier in pCDFDuet-1 plasmid. T7 promoter, the gene downstream of this promoter will be transcribed when there is T7 RNA polymerase. lacO, the sequence represses the nearby promoter when there is NO inducer (e.g. IPTG). RBS, ribosome binding site. hrpR, hrpS, the activator proteins. P_hrpL, a promoter which can be induced by the ultrasensitive high-order co-complex hrpRS. GFP, green fluorescent protein.

Figure 3. Responses of the GFP without fixed-gain amplification (V-GFP) and with fixed-gain amplification (V-AM). The cells are induced by 5 varying concentrations of IPTG (0, 10^-6 M, 10^-5 M, 10^-4 M, 10^-3 M) after 4 and 6 hours (A, 4h; B, 6h).

Method

We transformed 3 different vectors into E. coli BL21 competent cell. Amplifier and GFP sequences were inserted into pcdfDuet-1 vectors which were called V-AM and V-GFP. Empty vectors with amplifier or GFP were named VE, VE-GFP and VE-AM. After vector transformation, we grow the transformed competent cells on the LB plates with streptomycin, once we got colonies, we cultured single colony in 5 ml LB medium overnight at 37^oC. Next day, we washed bacteria by 1ml PBS and measured the cell growth pattern by photocytometer. In order to read the fluorescent signals, we used LB-S (LB with streptomycin) to dilute bacteria and added them into the black 96-well plate (Cat#: 3916, Corning Corp.) with OD₆₀₀=0.025 per well. Five different Isopropyl-beta-D-thiogalactopyranoside (IPTG) concentrations (0, 10^-3 M, 10^-4 M, 10^-5 M and 10^-6 M) were used to induce the T7 promoter activation and induced for 4 or 6 hours. After IPTG induction, the Enzyme Labeling Instrument were used to detect the green fluorescent signals. The excitation wavelength was 485 nm with the absorption wavelength 525 nm.

In conclusion, the amplifier achieves the desired effect, and the amplification gain is about 2 times. Comparing other amplifiers, this value is still low. In addition, we find that the gene expression process is accelerated with the amplifier, the reporter GFP usually reaching its saturation value after about 4 hours' induction.

Tuner

For the reducing regulation when the inflammation was not so serious, we designed another tunable-gain amplifier with the inhibitor protein, HrpV(Fig. 8).

Figure 8. Diagram for tunable-gain amplifier in pCDFDuet-1. As similar to fixed-gain amplifier in pCDF-duet1 plasmid, this system included PBAD promoter that was enabled to activate hrpV transcription and inhibit the hrpS effector protein function.

To find and show a clear result of tunable amplifier, T7 promoter would be replaced by the constitutive promoter J23109. Unfortunately, we have not constructed this part with the pCDFDuet-1 plasmid during this iGEM year. Our experiment has stalled in the extraction of plasmid DNA, even attempting too many times. After communicating with our adviser and consulting with the synthetic companies, the plasmid might not be suitable for such a part. We tried to construct it on other plasmids. But for the time limitation we built the Tuner Model to modify the tunable-amplifier in our “gut firemen” instead. (details about the Model, please click [http://2019.iGEM.org/Team:NEU_CHINA/Model here])

Reference

[1]Jovanovic,M., James,E.H., Burrows,P.C., Rego,F.G.M., Buck,M. and Schumacher,J. (2011) Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity. Nat. Commun., 2:177.

NEU_CHINA 2020

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 Sigmas, Anti-Sigmas, and Promoters." Mol Syst Biol 9 (2013): 702.

[2] Xinyi Wan et al. "Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals". 15.5(2019):540-548.

[3] Jesus, Fernandez Rodriguez , and C. A. Voigt . "Post-translational control of genetic circuits using Potyvirus proteases." Nuclc Acids Research 13(2016):6493-6502.

Sequence and Features