Part:BBa_K3611009

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 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.

Characterization by 2021iGEM_NOFLS_YZ

Improvement of an existing part

The group NEU_CHINA 2020 designed a composite part BBa_K3611009. They connected a secondary amplifier in series on the basis of the original hrp amplifier. The extracytoplasmic functional (ECF) sigma factor ECF11_987 and promoter ECF11_3726, which were proved as a higher-gain amplifier combination, were selected to build secondary amplifier.

Our team design the new composite part BBa_K3994011, with sequence different from the old one BBa_K3611009 (Figure 17). When and only when S2O32-/S4O62- and NO3- are present at the same time, the fluorescent gene is expressed. The fluorescent gene can be replaced with a gene that produce the interleukin-10 (anti-inflammatory factor). Our new composite part BBa_K3994011 was given a completely new function, producing the interleukin-10 (anti-inflammatory factor) for the treatment of IBD.

Figure 17 The blast results about the sequence of our new part BBa_K3994011 and the old one BBa_K3611009...

In addition, our project can serve General Public, Hospital and Biotechnology Firms lots of industrial biofactory companies. If our IBD biosensor may enter the market, it is possible to distribute it to ordinary people who want to have a test of IBD at home. IBD diagnosis has been a challenge for hospital because of its complex and various clinical symptoms. Thus, it should always be good to equip doctors with additional methods to help them better evaluate patients’ IBD related symptoms, which may help doctors enhance their diagnosis efficiency and efficacy. If we treat our development technique as a product, it is also possible to identify biotechnology firms as our potential clients. The biotechnology firms may be willing to buy our techniques and make further optimization

Usage and Biology

Background

Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory disease of unknown etiology, including ulcerative colitis (CD) and Crohn’s disease (CD). This chronic disease, which is prone to repeated deterioration, currently lacks unified diagnostic and treatment standards, and is posing a great threat to public health. Drug therapy (anti-inflammatory drugs) is the preferred treatment for IBD. However, studies in the past 10 years have found that 30-50% of IBD patients do not respond to anti-TNF treatment. In addition, after long-term use of anti-inflammatory drugs, the patient's intestinal microbial status changes over time, and the effect may be lost due to drug resistance. Therefore, we need to seek help from other treatments for IBD.

Construct design

We can build Boolean logic circuits to combine thiosulfate/tetrasulfate and nitrate biosensors to optimize performance, improve sensor accuracy and enhance robustness, and optimize intestinal inflammation detection (Figure 1). When and only when S2O32-/S4O62- and NO3- are present at the same time, the fluorescent gene is expressed (Figure 1). The fluorescent gene can be replaced with a gene that produce the interleukin-10 (anti-inflammatory factor) for the treatment of IBD. HrpS is a key functional factor under PttB344 promoter. amilGFP is a fluorescent protein under PhrpL promoter. IL10 is an anti-inflammatory factor.

Figure 1. Boolean gate logic circuit/AND circuit construction...

 Part1: PyeaR_HrpR, sensing NO3- then release the substance R;

 Part2: PttB344_HrpS_PJ23105_ttrR, sensing S4O62- then release the substance S;

 Part3: PhrpL_amilGFP_IL10_PJ23104_ttrS, will release fluorescence if sensing both the substances, R and S released from Part 1(HrpR) and Part 2 (HrpS) , respectively.

Figure 2. A. PyeaR_HrpR, sensing NO3- to release the substance R; B. PttB344_HrpS_PJ23105_ttrR, sensing S4O62- to release the substance S; C. PhrpL_amilGFP_IL10_PJ23104_ttrS...

The profiles of every basic part are as follows:

Usage and Biology

This codes for HrpS protein. HrpR protein binds to HrpS protein forming a complex and then triggering the transcription of promoter hrpL. This functions like a AND logic gate.

Usage and Biology

This is a sequence of PttB344. PttrB185-269 is a minimal TtrR activated promoter when TtrR is phosphorylated by TtrS after TtrS sensing tetrathionate.

Usage and Biology

It is the sequence of inducible promoter PhrpL. HrpR protein binds to HrpS protein forming a complex and then triggering the transcription of induced promoter PhrpL. It functions like a AND logic gate to open a gene circuit. The hrpL promoter (PhrpL) contains a functional UAS relative to the transcription start-site, to which purified HrpR, HrpS and HrpRS bind in electrophoretic mobility shift assays.

Usage and Biology

This is a coding sequence of IL10. This is a major immune regulatory cytokine that acts on many cells of the immune system where it has profound anti-inflammatory functions, limiting excessive tissue disruption caused by inflammation.

Experimental approach

Recombinant Plasmid Construction

Figure 3. The electrophoresis results of enzyme digestion and PCR...

Lane 1 and 2: Plasmid pSU2718-p15A digested by Xba1 and BamH1.

Lane 3: Part 2, PttB344_HrpS_PJ23105_ttrR, got by PCR method with size of 2060bp.

This step is used to get the plasmids pSU2718-p15A digested by enzyme Xba1 and BamH1and gene PttB344_HrpS_PJ23105_ttrR by PCR method for later in the process. Therefore, channel 1 and 2 were plasmids pSU2718-p15A digested by enzyme Xba1 and BamH1. And channel 3 were gene PttB344_HrpS_PJ23105_ttrR got by PCR. Clean-up the product to obtain pSU2718-p15A backbone and PttB344_HrpS_PJ23105_ttrR-fragment. T4 DNA ligase is used to connect pSU2718-p15A backbone and PttB344_HrpS_PJ23105_ttrR-fragment to get plasmid pSU2718-part2.

Figure 4 E-coil having the desired pSU2718-part2 (Left) and control (Right)...

Figure 5. The electrophoresis result of enzyme digestion identification...

Lane 1: Plasmid pSU2718-p15A digested by EcoR1.

Lane 2 to 5: Recombinant plasmid pSU2718-part2 digested by EcoRI. We got two bands with size of 2803bp and 1544bp.

The results show that we got the correct plasmid. And the plasmid was sent to sequence.

Figure 6 The result of sequencing for plasmid pSU2718-part2...

Sequencing feedback shows we have obtained the correct plasmids which is consistent with their DNA profiles.

Figure 7 The electrophoresis results of enzyme digestion (Middle) and PCR (Right)...

Middle:

Lane 1 and 2: Plasmid pSU2718-part2 digested by HindIII.

Right:

Lane 1: Part 1, PyeaR_HrpR, got by PCR method with size of 1293bp. This step is used to get the plasmids pSU2718-part2 digested by enzyme and gene PyeaR_HrpR by PCR method for later in the process. Therefore, channel 1 and 2 (Middle) were plasmids pSU2718--part2 digested by enzyme HindIII. And channel 1 (Right) was gene PyeaR_HrpR got by PCR. Clean-up the product to obtain pSU2718-part2 backbone and PyeaR_HrpR-fragment. T4 DNA ligase is used to connect pSU2718-part2 backbone and PyeaR_HrpR-fragment to get plasmid pSU2718-part2-part1.

Figure 8 E-coil having the desired pSU2718-part2-part1 (Left) and control (Right)...

Figure 9 The electrophoresis result of enzyme digestion...

Lane 1: Recombinant plasmid pSU2718-part2-part1 without enzyme digestion.

Lane 2: Recombinant plasmid pSU2718-part2-part1 digested by EcoR1.

The result shows that we got the correct plasmid. And the plasmid was sent to sequence.

Figure 10 The result of sequencing for plasmid pSU2718-part2-part1...

Sequencing feedback shows we have obtained the correct plasmids which is consistent with their DNA profiles.

Proof of function

Attempt 1

Figure 11 Genetic Construction of IBD Distinguisher...

Sample 1: NO3- + S4O62-

Sample 2: S4O62-

Sample 3: NO3-

Sample 4: Blank Control

As shown above, there is obvious fluorescence in sample 1 and sample 2; for sample 3 and sample 4, there also a slight fluorescence produced in the tubes. In order to scientifically quantify the result, we used ELIASA to read the accurate fluorescence intensity, respectively.

Table 1...

Figure 12. Histogram of the fluorescence intensity of samples in Table 1...

According to the histogram above, we can see that Sample 1 (NO3-+ S4O62-) presents obvious higher fluorescence intensity than the rest three samples which means our design working. But we do realize that Sample 2 (S4O62-) also presents fluorescence than the blank control which confused us a lot. According to our design, if we only add S4O62- without NO3-, this AND gate wouldn’t allow to pass into the reporter (Part 3) as there is a closed door of the NO3- sensor (Part 1). Therefore, we thought there was something wrong in Part 1.

In order to figure out the reason, we did several literature research and find out that in Part 1 we didn’t build the NarX-NarL two-component regulator (Figure 13), which shall be the first one to sense nitrate and that is why Part 1 didn’t work fine as we expected.

Figure 13. Genetic construction design map...

Ref. Woo, Seung-Gyun, et al. "A designed whole-cell biosensor for live diagnosis of gut inflammation through nitrate sensing." Biosensors and Bioelectronics 168 (2020): 112523.

Attempt 2

Figure 14...

Sample 1: Negative Control (E. coli)

Sample 2: Positive Control (E. coli/amilGFP)

Sample 3: E. coli/pUC-57_Part 3

Sample 4: E.coli/pSU2718-Part 1_Part 2+pUC-57_Part 3

In order to further analyze the performance of Part 2, we also designed the control groups as showing above where Sample 1 and Sample 2 were presented as the comparison of the fluorescence phenomenon. We can see that there was no fluorescence in Sample 3 (the report part, Part 3). According to the result of Sample 3 and Sample 4 (Figure 14), we could infer that the AND gate design works (HrpR_HrpS, PhrpL) as the Part 3 didn’t respond when there is no Part 1 and Part 2. Of course, we also repeated the experiments and measured the fluorescence intensity by ELIASA. The data is given below.

Table 2...

Figure 15. Histograms of the fluorescence intensity of samples in Table 2...

Figure 16. Trend Contrast of the fluorescence intensity between that under OD600=0.8 and that under OD600=1...

According to the trend in Figure 16, the fluorescence intensity of pUC57-Part 3 presents the similar level as that of blank control and group NO3-, which means the “gate keeper” promoter, PhrpL works well and this conclusion also back up the conclusion that the AND gate design works well (HrpR_HrpS, PhrpL).

As the group S4O62 did present higher fluorescence intensity than that of group NO3- whenever OD600 equals to 0.8 or 1, it indicates that Part 2 and Part 3 both works well as Part 2 did show “green light” to the thiosulfate to finally “light” the Part 3.

Future plan

So far, we have ensured that Part 2, Part 3 and also the AND gate design work, we need to improve the design of Part 1 which is used to sense nitrate so that we could fully complete the whole biosensor construction to diagnose IBD. Besides, we also have concerns about the biosensor carrier, E. coli. Firstly, we need ensure that our engineered bacteria would not be immediately degraded or eliminated by other enteric microorganism. Secondly, E.coli may have adverse effects such as septicemia in patients with immune deficiency to the gut microbiome. In the future, safer bacteria like lactobacillus should be found as carrier receptors for transformation.

References

4. Wang, Baojun, et al. "Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology." Nature communications 2.1 (2011): 1-9.

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