Team:BEAS China/Design
Summary of Project Design
NEZHA is a platform to detect and adsorb heavy metals through modular approaches. For the real-world applications, we also design an ncAA-dependent toxin-antitoxin biocontainment system in NEZHA.
In order to demonstrate the performance of NEZHA, we used Hg2+ in our project to test the functions of Sensor, Amplifier, Adsorption, and Biocontainment modules. Moreover, NEZHA is not a platform only to detect and adsorb Hg2+, but also can be employed to treat with other kinds of heavy metal contamination, which could improve the application value of the NEZHA platform greatly.
A. Basic Biosensor for Heavy Metal Detection
A basic biosensor for heavy metal detection consists of two major components. One part is a sensor, which is usually a transcriptional repressor. The other part works as a reporter, whose expression is under control of the sensor mentioned before. (See Figure 1)
When there is no or low concentration of heavy metal ions, the heavy metal sensing protein will bind to PR promoter, inhibiting the expression of the downstream output signal. When the concentration of heavy metal ions exceeds a certain threshold, the heavy metal sensing protein will interact with ion and then dissociates from the PR promoter, permitting the expression of the output signal.
Figure 1: The scheme of basic sensor design.
Based this principle, we designed the Basic Sensor for Hg2+ based on the BBa_K346001 and BBa_K346002.(See Figure 2A) Through model, we found that “The weaker the promoter (that is, the lower the MerR receptor concentration), the more sensitive and higher the dynamic range of the sensor”(See Figure 2B)". Click on the link to view our models and demonstrations.
Figure 2: A Circuit design of Mercury basic sensor B Modelling equations of the Hg basic sensor
B. Amplifier: amplify output signal for Heavy Metal Detection
To further improve the detection performance of the Sensor, we tried to insert an amplifier module based on the Basic Sensor to enhance the output signal. Mainly three designs are tested. B1: RinA_p80α activator; B2: TEV-C1434; B3: RinA_p80α-TEV-tevS-AAV Tag
B1: Amplifier 1 (RinA_p80α)
We worked with QHFZ to complete the design of Amplifier 1. In this design, Hg ions directly control the expression of RinA_p80α activator, and the activation promoter PRinA_p80α of RinA_p80α activator controls the output GFP. When Hg ions activate the expression of RinA_p80α activator, RinA_p80α activator will activate GFP expression on high-copy promoters, leading to a higher level of fluorescence values.
Figure 3: Circuit design of Mercury Amplifier using RinA_p80α
B2: Amplifier 2 (TEV-C1434)
We worked with UCAS_China to complete the design of Amplifier 2. In this design, Hg ions regulate the expression of TEV protease; the recognition site of TEV protease is inserted into C1434. In the presence of TEV protease, C1434 protein is cleavaged by TEV and loses its activity. In the absence of TEV protease, C1434 binds to the pR promoter and inhibits GFP expression at the transcriptional level. The final logic is that Hg2+ induces GFP expression. Due to the sensitivity of TEV itself, a small amount of expressed TEV protease can inactivate C1434, thereby allowing GFP expression.
Figure 4: A The scheme of Mercury Amplifier using TEV-C1434 B Circuit design of Hg Amplifier using TEV-C1434
B3: Amplifier 3 (RinA_p80α-TEV-tevS-AAV Tag)
The optimized third design is still based on the RinA_p80α transcriptional activation system. We added the TEV-tevS-AAV Tag system to the system. In this amplifier, the main TEV and RinA_p80α are regulated by Hg2+. We also fused the GFP protein with the tevS-AAV tag module. There are three factors control the expression of GFP: First, RinA_p80α regulates GFP at the transcriptional level; second, endogenous proteases of E. coli regulate GFP-tevS-AAV Tag. Once recognized, endogenous proteasesn degrade GFP-tevS-AAV Tag and eliminate fluorescence uotput; third, TEV protease can recognize tevS then cleavages the AAV Tag so that endogenous proteases will no longer affect the fluorescence level of the system.
Figure 5: Circuit design of Mercury Amplifier 3 (RinA_p80α-TEV-tevS-AAV Tag)
As can be seen from Figure 6A & B, the yellow line represents the GFP-tevS-AAV Tag expression level, the blue line represents the expression of TEV, and the red line is the actual fluorescence output signal. When TEV is not expressed, GFP-tevS-AAV Tag is degraded by endogenous proteases, which reduces the actual fluorescence output, alleviating the leakage of the Sensor; when TEV is gradually expressed, the GFP-tevS-AAV Tag is gradually cleavaged by TEV, making the fluorescence accumulate to higher level. Figure B shows sifferent TEV expression scenarios lead to different output.
Figure 6: A Different gene expression levels in Amplifier 3 (RinA_p80α-TEV-tevS-AAV Tag) B Different TEV expression scenarios lead to different output
C. Adsorption：Heavy Metal Adsorber
We hope to use cells as heavy metal adsorption machines and achieve modularization of heavy metal adsorption, therefore developing a more flexible system to adsorb different types of heavy metals without changing the basic design. Our design principles are as follows:
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We engineered A cells into the Docking Cell, which has a large amount of spytag docking sites on its cell surface that can be used to fix heavy metal adsorption proteins.
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We selected csgA and INP proteins for surface display of spytag, which further expanded the usage of available extracellular surface space. (To enable high efficiency of csgA surface display, we deleted csgA gene in C321 using CRISPR/Cas9)
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B cells are designed to express SpyCatcher-MBP only. Therefore, by just lysing B cells and adding the supernatant extract of proteins to the A cell culture medium(containing different heavy metal ions), we are able to process water with differnent heavy metal ions in a modular and flexible way.
C1: Spytag + SpyCatcher
The SpyTag/SpyCatcher system is a technology for the irreversible conjugation of recombinant proteins. The peptide SpyTag (13 amino acids) spontaneously reacts with the protein SpyCatcher (12.3 kDa) to form an intermolecular isopeptide bond between the pair. DNA sequence encoding either SpyTag or SpyCatcher can be fused to the DNA sequence encoding a protein of interest, forming a fusion protein. These fusion proteins can be covalently linked when mixed in a reaction through the SpyTag/SpyCatcher system.
Figure 7: The SpyTag/SpyCatcher system
C2: INP + csgA
Bacterial ice-nucleation proteins are a family of proteins that enable Gram-negative bacteria to promote nucleation of ice at relatively high temperatures (above -5°C). These proteins are localized at the surface of the outer membrane and can cause frost damage to many plants.
The CsgA protein is the major subunit of curli; it is actively secreted to the extracellular milieu, where CsgA monomers self-assemble into curli .
Figure 8: A INP surface dispaly B csgA surfave display
C3: Heavy Metal Adsorber
In the final version of NEZHA Heavy Metal Adsorber, we selected csgA and INP proteins to display spytag proteins on the extracellular surface space, which further expanded the usage of available extracellular surface space. We also engineered B cells to express SpyCacther-MBP specifically. Therefore, by just lysing B cells and adding the supernatant extract of proteins to the A cell culture medium(containing different heavy metal ions), we are able to process water with heavy metal ions in a modular and flexible way.
Figure 9: Heavy Metal Adsorber A & B Cell
We have also designed the appropriate hardware for the heavy metal adsorption process. See Hardware.
Figure 10: A NEZHA Adsorber B NEZHA Hardware
D. Biocontainment: GMO In Lockdown
Synthetic Biology is a growing field which holds great promise in several areas of application. However, it also poses serious risks for human health and the environment that must be anticipated in order to develop effective prevention and management measures.
In order solve this cirtical problems, we need a biocontainment system that could restrict NEZHA in a defined environment. Our design is based on two main parts:
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Part I: Toxin-Antitoxin system. There is a wide range of TA systems in nature. The expression of Toxin will lead to cell death, while Antitoxin can neutralize Toxin to ensure the normal growth of cells.
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Part II: Non-canonical amino acid system (ncAA). ncAA are synthetic molecules, which doesn't in nature. By making the survival of bacteria depend on unnatural amino acids, we can avoid the drawbacks of the traditional Biocontainment system. The non-natural amino acid system has been widely used in bio-security prevention and control systems, with well performance and the escape rate lower than 10-13.
Figure 11: GMO In Lockdown
D1: Toxin-Antitoxin (PhD-Doc)
Doc toxin:Toxic component of a type II toxin-antitoxin (TA) system. Overexpression of Doc results in inhibition of growth in liquid cultures and a decrease in colony formation by inhibiting translation, stabilizing mRNA and polysomes; these effects are overcome by concomitant expression of antitoxin phd. DOc binds 70S ribosomes and the 30S ribosomal subunits, the binding site is the same as for the antibiotic hygromycin B[1].
Phd antitoxin: Antitoxin component of a type II toxin-antitoxin (TA) system. Phd is a labile antitoxin that binds to cognate doc toxin and neutralizes its ability to phosphorylate host EF-Tu. [1]
The phd/doc family is one the smallest families of toxin–antitoxin modules and was first discovered as a plasmid addiction module on E. coli bacteriophage P1. phd and doc proteins function in unison to stabilize plasmid number by inducing a lethal response to P1 plasmid prophage loss.[2]
D2: ncAA System
In protein translation, an aminoacyl-tRNA synthetase (aaRS) loads its cognate tRNA with a specific amino acid. Then, the tRNA is pulled into the ribosome and if the anticodon on the tRNA can bind to the mRNA (hence, the anticodon is complementary to the codon), the amino acid from the tRNA is incorporated into the growing peptide chain.
Figure 12: ncAA system compared with cononical anmino acid system
To expand the genetic code, modified tRNAs, codons, and tRNA synthetases are introduced into the cell on plasmids and the new amino acid is introduced in the media.
Figure 13: Schematic illustration of genetic circuits for incorporation of 2 ncAAs into GFP(39TAG & 151TAG).
D3: E.coli C321
E.coli C321 is recoded E. coli MG1655 strain with UAG termination function removed (RF1 is deleted). E.coli C321 is commonly used as the chassis for non-canonical amino acid incorporation into proteins.
Figure 12: ncAA system compared with cononical anmino acid system
To expand the genetic code, modified tRNAs, codons, and tRNA synthetases are introduced into the cell on plasmids and the new amino acid is introduced in the media.
Figure 13: Schematic illustration of genetic circuits for incorporation of 2 ncAAs into GFP(39TAG & 151TAG).
D4: ncAA-dependent Toxin-Antitoxin system for biocontainment
Finally, in order to couple the survival of bacteria with ncAA, we tried to build a toxin-antitoxin system dependent on ncAA. Such a system has two advantages. First, Doc toxic protein can effectively lower the escape rate of bacteria. Second, due to the absence of ncAA in nature, Metabolite crossfeeding between different bacterial groups could be avoided.
Figure 14: ncAA-dependent Toxin-Antitoxin system for biocontainment
References
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Liu M, Zhang Y, Inouye M, et al. Bacterial addiction module toxin Doc inhibits translation elongation through its association with the 30S ribosomal subunit[J]. Proceedings of the National Academy of Sciences, 2008, 105(15): 5885-5890.
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Lehnherr H, Maguin E, Jafri S, et al. Plasmid addiction genes of bacteriophage P1: doc, which causes cell death on curing of prophage, and phd, which prevents host death when prophage is retained[J]. Journal of molecular biology, 1993, 233(3): 414-428.
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Liu X, Jiang L, Li J, et al. Significant expansion of fluorescent protein sensing ability through the genetic incorporation of superior photo-induced electron-transfer quenchers[J]. Journal of the American Chemical Society, 2014, 136(38): 13094-13097.
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Fankang, Meng, and Lou Chunbo. "Research Progress in Biocontainment of Genetically Modified Organisms." Chinese Journal of Organic Chemistry 38.9 (2018): 2231-2242.
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Zheng, Yang, et al. "A tight cold-inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts." Nucleic Acids Research (2019).
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Lajoie MJ, Rovner AJ, Goodman DB, Aerni HR, Haimovich AD, Kuznetsov G, Mercer JA, Wang HH, Carr PA, Mosberg JA, Rohland N, Schultz PG, Jacobson JM, Rinehart J, Church GM, Isaacs FJ. Science. 2013 Oct 18;342(6156):357-60. doi: 10.1126/science.1241459. 10.1126/science.1241459