![](https://static.igem.org/mediawiki/2017/6/66/T--Jilin_China--_sec_bg_mr.png)
Water pollution is a serious problem all over the world. It takes a lot to handle with sewage every year. There are 3 mostly used methods of sewage treatment:
- Physical processing: separate and collect water-insoluble impurities in waste water.
- Chemical treatment: separate or transform some kind of solutes and colloidal particles into non-hazard chemicals.
- Biological metabolism: transform some kind of solutes and colloidal particles into stable non-hazard substances.
Biological treatment is one kind of high efficiency and environment friendly method to purify sewage. Since many species of microorganisms could hardly be separated from natural environment and cultured in artificial condition, main focus is put on engineered microorganism.
Ⅱ. Circuit
DmpR and Toxin are downstream of a constitutive promoter, antitoxin and Enzyme are downstream of pdmp operon (Fig 1). When there are no phenolic components in environment, DmpR protein can be ready to activate dmp operon and toxin can be expressed to repress growth of our engineered bacteria. When aromatic substrates appear, DmpR protein can combine pdmp and trigger antitoxin and enzyme expression, leading to toxin neutralization and phenolic degradation, respectively.
![](https://static.igem.org/mediawiki/2017/8/8a/T--Jilin_China--application01.png)
Figure 1. The design of whole circuit.
Key achievements
1.Comparing different promoting strength of several promoters.
2.Picking out promoter with applicable promoting strength.
Introduction
![](https://static.igem.org/mediawiki/2017/3/3b/T--Jilin_China--application02.png)
Figure 2. Circuit for sensor function certification
Results
1.J23101 showed strongest promoting activity.
To figure out whether EGFP expression will have effects on bacteria growth, we tested the growth rate of different constructed bacteria containing different promoters. The growing tendency of bacteria expressing EGFP was similar with unloaded vector within 12 hours, which suggested that in 12 hours, the expression of EGFP wouldn’t have effects on the growing of bacteria.
We used the ratio of EGFP and value of OD600 to make comparisons on promoter strength. It turned out that promotor J23101 expressed strongest among all the promotors, and J23107 next. Except for these two promotors, others showed no difference with mock in expression level.
![](https://static.igem.org/mediawiki/2017/1/16/T--Jilin_China--application03.png)
![](https://static.igem.org/mediawiki/2017/9/95/T--Jilin_China--application04.png)
Figure 3. Promoter strength certification
(A) Growth curve of different constructed bacteria. Constructions contained promoter J23101, J23107, J23114, wt-Pr and mock (B) ratio of EGFP/ OD600
2.DmpR downstream of strongest promoter had the greatest sensitivity
To probe whether promoter strength will influence DmpR’s sensitivity toward phenolic pollutions, we chose weak promoter J23114, medium promoter J23107 and strong promoter J23101. Phenol, 2-CP, 4-CP and pyrocatechol are selected to test DmpR’s sensitivity. Comparisons are based on ratio of different promotors’ response and mock’s response to same phenolic component respectively.
Among three promotors, DmpR downstream of J23107 responded strongest towards phenol, 4-CP and pyrocatechol. Under J23107, DmpR’s response towards phenol was about 1.5 times of pyrocatechol's, and about eight times than 4-CP’s. under J23101, its response towards phenol was lower than pyrocatechol’s, in accordance with J23114. Besides, promotor J23101 and J23107 didn’t show any response to 2-CP. To sum up, promotor J23107 acts best in the response to phenolics.
![](https://static.igem.org/mediawiki/2017/6/6d/T--Jilin_China--application05.png)
Figure 4. response intensity of DmpR downstream of promoter with different strength
Key achievements
1. Overexpression and purification of monooxygenase TfdB-JLU
2. Determination of the substrate preference and activity of TfdB-JLU in vitro
3. Certification of TfdB-JLU catalytic activity in vivo
Introduction
![](https://static.igem.org/mediawiki/2017/b/b8/T--Jilin_China--application06.png)
Figure 5. Initial two steps in phenol degradation
Usually, monooxygenases have harrow substract preference. However, this year, the monooxygenase we use is TfdB-JLU, a novel 2,4-dichlorophenol hydroxylase whose amino acid sequence exhibits less than 48% homology with other known TfdBs[5,6]. Compared to wildtype TfdB, TfdB-JLU has a wilder substrate range and higher catalysis activity. To induce expression of tfdB-JLU, we constructed pET28a-JLU expression vector and transformed it into BL21(DE3)-pLysS.
Because substract and production of tfdB-JLU share close maximum absorbance, along with tfdB-JLU is a NADPH dependent enzyme, we determined enzyme activity by monitoring the decrease in absorbance at 340 nm (e340 = 6,220 M-1 cm-1) following the substrate-dependent oxidation of NADPH.
To certify in vivo activity, we use 4-AAP assay. Phenol can react with 4-AAP in alkaline medium (pH =10.0±0.2), with the oxidizer K3Fe(CN)6. Reaction product antipyrine dye appears orange, whose absorption peak is at 510nm.
![](https://static.igem.org/mediawiki/2017/3/3d/T--Jilin_China--application07.png)
Figure 6. Mechanism of 4-AAP assay
pET28a-tfdB-JLU and mock (unloaded pET28a) vector were transformed into BL21. Then digestion assay and sequencing were done to confirm. Several experiments were designed to certify TfdB-JLU’s function, including in vitro and in vivo assay.
1.Overexpression and purification of TfdB-JLU
2.Enzyme assay in vitro
3.Enzyme assay in vivo
1. Overexpression and purification of TfdB-JLU
1.1 Digestion assay
![](https://static.igem.org/mediawiki/2017/4/46/T--Jilin_China--application08.png)
Figure 7. Digestion assay of pET28a-tfdB-JLU
1.2 Overexpression and purification of TfdB-JLU
![]() |
Purified TfdB-JLU was eluted in 250mM imidazole elution buffer (lane 7, lane 8) Line 1: crude extract Line 2: flow through Line 3: 20 mM imidazole washing buffer Line 4: 50 mM imidazole washing buffer Line 5, 6: 100 mM imidazole washing buffer Line 7, 8: 250 mM imidazole elution buffer Line 9: 500 mM imidazole buffer |
1.3 substrate activity and preference of TfdB-JLU
substrate | Relative activity with FAD (%) | Specific activity (U/mg) |
---|---|---|
3-CP | 311 | 1.9144 |
4-CP | 37 | 0.2295 |
Phenol | 75 | 0.4590 |
2,3-DCP | 110 | 0.6717 |
2,4-DCP | 100 | 0.6157 |
2,5-DCP | 55 | 0.3470 |
2,6-DCP | 143 | 0.8788 |
3,4-DCP | 16 | 0.1008 |
3,5-DCP | 17 | 0.1064 |
2,4,5-TCP | 31 | 0.1903 |
For determining substrate specificity, the enzyme was incubated in 50 mM sodium phosphate buffer, pH 7.5, with 0.1 mM substrate under standard conditions. Relative activity is expressed as a percentage of the maximum enzyme activity towards 2,4-DCP with FAD.
2. Phenol degradation assay in vivo
Among phenolic components, methods in phenol detection are easiest and widely used. Thus, we chose phenol as the substract for degradation experiments in vivo. We used two methods, one is 4-AAP assay, in which phenol can react with 4-AAP in alkaline medium (pH =10.0±0.2), with the oxidizer K3Fe(CN)6. Reaction product antipyrine dye appears orange, whose absorption peak is at 510nm. The other one is HPLC.
2.1 HPLC
![](https://static.igem.org/mediawiki/2017/d/dd/T--Jilin_China--application10.png)
![](https://static.igem.org/mediawiki/2017/5/51/T--Jilin_China--application11.png)
Figure 9. HPLC analysis of phenol degradation Phenol-PBS solution (A) before incubation and (B) after incubation with engineered bacteria
![](https://static.igem.org/mediawiki/2017/e/eb/T--Jilin_China--application12.jpg)
Figure 10. Phenol degradation potency in vivo
2.2 4-AAP assay
![](https://static.igem.org/mediawiki/2017/6/69/T--Jilin_China--application13.jpg)
![](https://static.igem.org/mediawiki/2017/e/ee/T--Jilin_China--application14.jpg)
Figure 11. 4-AAP analysis of phenol
(A) assay system without K3Fe(CN)6 (+ 4-AAP + phenol)
(B) assay system with K3Fe(CN)6 (+4-AAP + phenol) Phenol concentration from left to right: 0mM、1mM、2mM、5mM
![](https://static.igem.org/mediawiki/2017/a/a3/T--Jilin_China--application15.png)
Figure 12. 4-AAP analysis for phenol degradation potency in vivo
We successfully certified TfdB-JLU can degrade phenolic components both in vivo and in vitro. But enzyme showed higher activity in vitro than in vivo. There are two possible reasons for this. Firstly, E.coli we use have a complicated intercellular environment, which can affect enzyme activity, also, the way phenol pass the cell wall is free diffusion, which may have lower efficiency in catalyzed by enzyme. Secondly, we found our medium appeared blue after 16h induction, this is because TfdB-JLU can produce indigo through tryptophan, which has indolyl. Thus, for phenol, tryptophan may cause competitive inhibition. The phenomenon that medium appeared blue can further prove TfdB-JLU can work in vivo.
Ⅴ. Future work
1. Move circuit into Bacillus or other bacteria used widely for phenolics disposing.
2. Artificially constructed an efficient enzyme system which have wide substract range for phenolics degradation.
3. Find a novel application for combination with sensor and enzyme like Industrial raw material production.
Reference:
[1] V. L. Campos, Detection of Chlorinated Phenols in Kraft Pulp Bleaching Effluents Using DmpR Mutant Strains. Bull. Environ. Contam. Toxicol. 2004 73:666–673
[2] V. L. Campos, Monitoring Phenolic Compounds During Biological Treatment of Kraft Pulp Mill Effluent Using Bacterial Biosensors. Bull. Environ. Contam. Toxicol. 2006 77:383–390
[3] ARLENE A. WISE. Generation of Novel Bacterial Regulatory Proteins That Detect Priority Pollutant Phenols. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 0099-2240/00/$04.0010 Jan. 2000, p. 163–169
[4] Huiqing Chong, Development of Colorimetric-Based Whole-Cell Biosensor for Organophosphorus Compounds by Engineering Transcription Regulator DmpR. ACS Synth. Biol. 2016, 5, 1290−1298
[5] Ledger T, Pieper DH, Gonzalez B, Chlorophenol hydroxylases encoded by plasmid pJP4 differentially contribute to chlorophenoxyacetic acid degradation. Appl Environ Microbiol 2006 72:2783–2792
[6] Yang Lu, Cloning and characterisation of a novel 2,4-dichlorophenol hydroxylase from a metagenomic library derived from polychlorinated biphenyl-contaminated soil. Biotechnol Lett 2011 33:1159–1167