Team:HiZJU-China/Design
Launching idea – rooted on Molecular Reaction Dynamics
There exist several kind of autotrophic ammonia oxidation that can produce ammonia oxidase strains. In recent years, the application of ammonia oxidizing bacteria to dispose waste containing nitrogen-related contaminants which is getting heated and become a big innovations and growth point in environmental protection topic. But because of the complicated mechanism of ammonia oxidation bacteria as biological chassis in the activated sludge system, A variety of complex exogenous environmental factors affect the growth and metabolism of AOB, in another words, affect its ability to co-metabolize and degrade EE2. Copper, iron, manganese, zinc, etc., are essential components of many metal enzymes of AOB, and have certain toxicity. The effect and mechanism of AOB co-metabolizing EE2 degradation by stress or reinforcement factor at low or high concentration is still unclear, which hinders the development of AOB to remove EE2 in wastewater treatment system. Comparably, heterotrophic bacteria used in the form of activated sludge sewage treatment technology have been widely used in the industry. After concentrating research on the total metabolic pathway of nitrogenous substances, we figure out these conclusions as followed:
1. When ammonia oxidizing bacteria degrade other organic amines, they need to maintain their normal growth on the basis of the existence of inorganic nitrogen sources in the environment, and obtain NADH required for growth and metabolism by oxidizing NH4+ in water through AmoA.
2. AmoA can oxidize organic amines apart from NH4+, which should be based on the normal growth of AOB, that is, the oxidation of organic amines requires the "activation" of NH4+. At the same time, they share the same reaction site and oxidation mechanism, and NH4+ is a competitive inhibitor of the oxidation of organic amines. NH4+ acts as a limiting factor for the oxidation of organic amines to some extent. That is the mechanism of AOB co-metabolizing EE2.
3. Ammonia-oxidizing bacteria generate NO by subsequent treatment of NH2-OH oxidized by AmoA through hydroxylammonium oxidoreductase HAO, which can reduce ammonia ion content in water to a certain extent. At the same time, the metabolism of AOB includes nitrification pathway, which can generate a certain amount of NO2- in water. NO2- degrades EE2 into other degradation products such as Nitro-EE2 through abiotic reaction process. Through LC50 analysis of EE2 and its transformation products, the toxicity of EE2 can be effectively reduced through biological metabolism process presenting in forms of EE2-OH. However, nitro-EE2 produced by non-biological metabolic processes is significantly more toxic than EE2, which may increase the harm to aquatic systems.
4. Compared with homogenized AOB bacteria, activated sludge with heterotrophic bacteria has more diversified and thorough types of transformation products. The reason may be that heterotrophic bacteria in sludge can degrade intermediates such as EE2-OH until mineralization, and the toxicity of the transformation products is educed step further.
Therefore, we came up with the idea of constructing EE2 degradation engineering bacteria by introducing AmoA and HAO genes from AOB bacteria into heterotrophic escherichia coli BL21. Combined with the effective oxidation of EE2 by amoA and the synergic degradation of NH4+ by AMOA-HAO, nitrogen components in wastewater were comprehensively treated. At the same time, the oxidation process of heterotrophic bacteria is utilized to avoid the degradation of EE2 due to insufficient NH4+ content, and ee2-OH is followed up to a certain extent. Meanwhile, the characteristics of high growth rate and environmental adaptability of heterotrophic bacteria also provide more possible directions for hardware design.
Experiment principles
Insert AmoA and HAO via pETDuet plasmid into engineering bacteria BL21. EE2 was degraded to EE2-OH through mono oxygenation reaction conducted by AmoA, and NH4+ in the reaction system was oxidized to separate from the system in forms of NO by AmoA and HAO to avoid the accumulation of NO2- in the solution.
Some studies have reported that ammoxidation bacteria can effectively degrade estrogen E1, E2, E3 and EE2.
Nitrosomonas Europaea ATCC 19718, an ammonia oxidizing bacteria, convert ammonia to hydroxylamine under the action of AMO and subsequently convert hydroxylamine to nitrite under the action of HAO.
Fig. 1-1
According to existing studies, the EE2 biotransformation obviously depends on the oxidation of NH3, that is, the degradation of EE2 needs to be carried out in the environment of NH3. The possible mechanism is that THE HAO oxidizes NH3 to provide electrons for the oxidation of EE2-OH. Ammonia is present in urine and sewage, so degradation can be achieved without the addition of other substances to the reaction system.
Fig. 1-2
We constructed peT-amoA-hao plasmid to introduce amoA and hao genes of ammonia oxidant into E.coli, and constructed the engineering bacteria we needed. When ammonia is present in sewage system or in urine environment, the recombinant Escherichia coli can degrade EE2.
At the same time, we designed a gene pathway controlled by the PBAD promoter in consideration of biosafety and preventing the leakage of engineered bacteria from harming the environment. The PBAD promoter is induced by arabinose. In the presence of arabinose, arabinose binds to AraC dimer and changes the conformation of the dimer. The PBAD promoter interacts with AraI1 and AraI2 manipulation gene sites to inhibit the transcription of downstream mazF toxin protein genes. In the absence of arabenose, the dimerization repressor araCs will bind to the AraI1 manipulation locus of PBAD, and the upstream manipulation locus AraC2 will initiate transcription of PBAD. In conclusion, in the presence of arabinose, the expression of toxin proteins is inhibited and the engineered bacteria can survive.
Promotion
1. In the early stage of experiment we get result that although the protein is produced but is lack of activity to degrade EE2. Thus we consult Prof.Lu, ZheJiang University for the question and finally make it clear that the amoA has several subunits, whose gene sequence we put in is incomplete so there's no biological activity. After going through related database we finally refer to the Aeromonas hydrophila isochorismate synthases AmoA sequence from NCBI database.
2. After successfully expressing the activity, we found the problems of shallow markers in SDS-PAGE and low activity of protein. After asking the technical personnel of gene synthesis company, we found that the metabolic systems of heterotrophic bacteria and autotrophic bacteria were different, so we adopted the method of optimizing codon and inducing expression to make the target gene protein expressed in large quantities.
3. Due to the high limit of detection when we detect EE2 by HPLC-hv , It happened that the background noise overshadowed the EE2 peak, after communicating with instructor Jiachang Lian. We expanded detection system from the original 10ml bacterial liquid system to 50ml bacterial liquid system by using the method of extraction and concentration, and obtained obvious HPLC peak of EE2 and its subsequent metabolites.
Fig. 1-1
Fig. 1-2
Currently, HPLC, GC surface and other traditional methods are used in EE2 detection. These methods require expensive equipment and demand sample processing. We hope to design a simple and portable bioassay system based on the knowledge of synthetic biology.
In our body, environmental estrogen signal transduction is achieved in a ligand-dependent manner. The ligand binding domain (LBD) of nER is recognized by estrogen-like hormone diffused into cells or estrogen synthesized in cells. Binding of estrogens to estrogen receptors causes a conformational change that allows the receptors to dissociate from an inhibitory complex of proteins, bind as dimers to specific regulatory sequences (enhancer elements) that are associated with the target genes regulated by estrogens. Subsequently, co-regulatory factors including co-activators and co-suppressors are recruited to promote or inhibit the binding of EEs-nER complex with downstream target gene promoter estrogen response element (ERE) to regulate gene transcription.
Fig.2-1 Estrogen signal transduction pathway
According to the estrogen signal transduction pathway, we decided to use yeast two-hybrid assay as the detection method of biological detection system, and red and green fluorescent protein GFP as indication.
The technical details of Yeast Two-Hybrid Assay are outlined in the cartoon below. Two fusions (‘hybrids’) are constructed between each protein of interest and either the DNA Binding Domain (DBD) or the Activation Domain (AD) of the TF. The protein fused to the DBD is referred to as the ‘bait’, and the protein fused to the AD as the ‘Prey’. Upon interaction between the bait and the prey, the DBD and AD are brought in close proximity and a functional TF is reconstituted upstream of the reporter gene.
Fig.2-2 The technical details of Yeast Two-Hybrid Assay
Estrogen nuclear receptor exists in a wide range of organisms and is expressed in sexual organs, lung, bone, heart, brain and other organs. There are two subtypes of human estrogen receptors (ERα and ERβ) that differ in tissue distribution. It has been reported that when transcription from a gene depends on both AF1 and AF2, the activity of ERα greatly exceeds that of ERβ. To order to enhance the detection ability of EE2, we choose ERα as biosensor.
First, we introduced red fluorescent protein RFP gene and green fluorescent protein GFP into saccharomyces cerevisiae Y2H to construction red fluorescent yeast Y2H-RFP and green fluorescent protein Y2H-GFP regulated by T7 promoter, respectively. The bait plasmid PGBKT7-ERα-LBD and the hunting plasmid PGADT7-Grip1 were constructed from the ligand binding domain of nER and glutamate receptor interacting protein, respectively. When EE2 exits, the complex formed by EE2-ERα and GRIP binds to the T7 promoter to initiate the expression of report genes. Red fluorescent protein RFP can be seen with naked eyes, while green fluorescence intensity needs to be quantitatively detected by multi-functional microplate meter or flow cytometer.
Fig.2-3 The Yeast Two-Hybrid Assay for detection of EE2
In order to lower the detection limit of the biological detection system, we modified the ligand binding domain (LBD) of estrogen receptor by artificial intelligence-assisted protein rational design to improve the specificity and affinity of receptor. According to the protein model result, we found the mutation M421F would have a good effect.
Fig.2-1 Estrogen signal transduction pathway
Fig.2-2 The technical details of Yeast Two-Hybrid Assay
Fig.2-3 The Yeast Two-Hybrid Assay for detection of EE2
We designed different kill switches for each part of our design to ensure bio-safety of products applied in different situation.
Launching idea – rooted on Molecular Reaction Dynamics
the engineered yeast in detection system is designed to work in a testing device, it would be convenient if the survival of yeast depends on a specific substance exists in the testing device.
At first, we envisioned a switch, with which, the engineered yeast can only survive when L-arabinose exists in the culture medium in the testing device.
But we found a more sophisticated kill switch designed by XMU-China in 2020. They introduced the inverter to achieve similar effect. The engineered bacteria will survive when the concentration of L-arabinose is high enough, but die when is low. L-arabinose is added into the culture medium in the device to ensure that the engineered bacteria could survive and work robustly. Once engineered bacteria escape to the external environment, the expression of mazF will cause the engineered bacteria die due to MazF protein's toxicity.
Fig.3-1 Gene circuit of kill switch for detection system
Kill switch for degradation system
For degradation system, the kill switch has similar mechanism but functions reversely.
In the absence of arabinose, the dimerization repressor protein AraCs will bind to the AraI1 operator site of PBAD and block the transcription of PBAD. When L-arabinose is added, AraC dimerization binds to it leading to conformational changes and thereby allowing downstream transcription of toxic protein MazF.
Fig.3-2
Targeting for sewage treatment plant and commercial use, we hope the engineered bacteria will die automatically when the EE2 is completely degraded. It struck us that negative-edge triggers in electrical circuits possess desired functions. We borrow this model and introduce negative edge triggered pulse detecting circuit shown in Fig.3(a). MazF is expressed only under the condition of both the existence as an input and subsequent elimination of EE2. The complex of EE2 and estrogen receptor (ER) binds to estrogen response element(ERE) in the promoter and initiate the expression of cIDN and cI.
CIDN is a mutant of CI repressor bearing a mutation in the DNA binding surface which eliminated the binding affinity of the cI mutant to operator sites but increased the affinity to non-specific DNA binding site. When the transcription starts, with a stronger RBS, many more CIDN molecules are present in the cell compared to CI molecules. Therefore, almost all of the monomers will form heterodimers with CIDN, and there will be no CI2 to activate PRM promoter. After the induction period, because of the degradation tag added with CIDN to ensure quick degradation of the monomeric proteins, CIDN molecules degrade quickly giving CI molecules a chance to form dimers and activate PRM promoter leading to the expression of mazF.
Fig.3-3
Fig.3-1 Gene circuit of kill switch for detection system
Fig.3-2
Fig.3-3
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