Team:NAU-CHINA/Proof

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Proof

Bioremediation is considered to be a major method of degrading contaminants in the environment.

We demonstrate five devices in our project. The five devices consist of the device to degrade 3-phenoxybenzoate, 3-hydroxybenzoate, catechol respectively and a device with the function of thoroughgoing degradation. In addition，we also assemble a device with mhbR, mhbpt promoter and reporter to realize 3-HBA induction in Escherichia coli.

1.Proof of 3-PBA degradation

Engineering bacteria harboring both pbaA1A2B and pbaC acquires the ability to degrade 3-phenoxybenzoate, producing equimolar amounts of 3-hydroxybenzoate and catechol.

PbaA1A2B are the oxygenase and ferredoxin components of the angular dioxygenase (3-phenoxybenzoate 1,2-dioxygenase) responsible for the angular dioxygenation at the 1' and 2' positions on the benzene moiety of 3-phenoxybenzoate and that the ferredoxin PbaB is indispensable for the angular dioxygenase. PbaC is a kind of reductase transferring electrons to the 3-phenoxybenzoate 1, 2-dioxygenase.(Fig.1A)

We used a strong constitutive promoter (BBa_J23101), to overexpress the gene cluster pbaA1A2B and C23O. An insulator (RiboJ) was applied to eliminate the impact of a part-junction sequence that was added by a promoter to the 5'-UTR. Constitutive promoter (BBa_J23100) was employed for to guarantee the steady transcription of gene pbaC. (Fig.1B)

Fig.1 A Gene cluster pbaA1A2B and pbaC can degrade 3-PBA, producing 3-hydroxybenzoate and catechol. B The device can degrade 3-PBA

When testing degradation of 3-PBA using engineering bacteria, we found that some of the solution getting brown for a while(Fig.2A). And the color it shows is the color of the oxide of catechol solution(Fig.2B). Although we have catechol 2，3- dioxygenase, some catechol may get into solution through cell membrane making the solution get brown.

Fig.2 A. Solutions to test degradation become brown for a while. B. α is the bottle with engineered bacteria and 1000μmol/L 3-PBA. Bottle β is the solution of Catechol which has been oxidized. The color of the two bottle is almost the same.

From analyzing degradation data, we found that about 10-20% of 3-PBA was degraded by our engineering bacteria within 60 hours. And most of the degradation happened in 24 hours.(Fig.3A) 3-PBA will restrain the growth of engineering bacteria a lot when its concentration is larger than 1000μmol/L.(Fig.3B) However, comparing with the growth situation of Sphingobium wenxiniaeJZ-1T in same concentration of 3-PBA ,our engineering bacteria can adapt to high concentration of 3-PBA and Sphingobium wenxiniae JZ-1^T couldn’t be tolerant of high concentration of 3-PBA.(Fig.3C)

Fig.3 A. Engineered bacteria`s degradation of 3-PBA B. 3-PBA`s restraint of our engineering bacteria. C Comparison of growth situation between JZ-1 and engineered bacteria

Discussion

We thought that catechol came out from the dead cell since cell let out its inclusion while dying. The darker the solution color is, the more catechol was excreted outside. Only the concentration of 3-PBA is equal to or larger than1000μmol/L can have enough catechol be produced by cells to change the color of solution(Fig.3A). Because of the restraint of 3-PBA, the bacteria grew slower as 3-PBA's concentration rising(Fig.3B). Therefore our engineered bacteria in solution with 1000μmol/L 3-PBA can produce a lot of catechol and let it out much more than other groups since more cells were dead than in solution with 1500μmol/L of 3-PBA. As a result, the middle bottle had the darkest color.(Fig.2A)

2.Degradation of 3-Hydroxybenzoate

MhbM, MhbD, MhbI, and MhbH converted 3-hydroxybenzoate to yield pyruvate and fumaratein.(Fig.4A)

In this device, the copA promoter is applied. Functional gene cluster mhbDHIM can be regulated by copper ions. Constitutive promoter (BBa_J23100) was employed to produce MhbT which is responsible for the uptake of 3-hydroxybenzoate.(Fig.4B)

Fig.4 3-hydroxybenzoate can be degraded by gene cluster MhbDHIM step by step B The device can degrade 3-hydroxybenzoate absolutely

We placed the device into DH5ɑ and tested its function, the result shows that the 3-HBA can be degraded thoroughly within 24 hours. And the concentration below 5000μmol/L can be degraded thoroughly within 12 hours.(Fig.5 A)

Fig.5 Engineering bacteria`s degradation of 3-HBA in different concentrations.

3.Proof of Catechol degradation

Catechol 2, 3-dioxygenase is the key enzyme for the biodegradation of catechol. It transforms catechol by meta-cleavage to 2-hydroxymuconate semialdehyde.(Fig.6A) We assembled a device which can degrade catechol effectively. Constitutive promoter (BBa_J23100) was applied to open the synthesis of Catechol 2,3-dioxygenase.(Fig.6B)

Fig.6 A. Catechol is degraded by Catechol 2, 3-dioxygenase, becoming 2-hydroxymuconate semialdehyde. B. The device can degrade catechol

We devided a plate into three areas, with each three streaked of three different colonies. "J23100 + B30034 + C23O + dT" and "J23100 + C23O(with native RBS) + dT"are colonies transformed with C23O coding gene, while negative control(CK) has none C23O gene. Before dripping catechol solution onto the colonies, the three streaks showed the same color colonies.(Fig.7A)After dripping 0.2mol/L catechol solution onto the colonies, the color of "J23100 + B30034 + C23O + dT" and "J23100 + C23O(native RBS) + dT" colonies turned to bright yellow(Fig.7B).Because catechol 2, 3-dioxygenase can be excreted outside, which suggests that our C23O gene is able to work. [2]

Fig.7 A Before dripping catechol solution onto the colonies, the three streaks showed the same color B After dripping 0.2mol/L catechol solution onto the colonies, the color of "J23100 + B30034 + C23O + dT" and "J23100 + C23O(native RBS) + dT" colonies turned to bright yellow.

4.Proof of thoroughgoing degradation

In this device, we used a strong constitutive promoter (BBa_J23101), to over express the gene cluster pbaA1A2B and C23O. Constitutive promoter (BBa_J23100) was employed for to guarantee the steady transcription of gene pbaC. The copA promoter is applied. Functional gene cluster mhbDHIM can be regulated by copper ions.(Fig.8A)

The whole system was placed on two different plasmids J61002and PCC1(Fig.8B),for J61002 and PCC1 have different antibiotic resistance .The gene pbaA1A2B and C23O were placed on PCC1 and the rest of genes were placed on plasmid J61002.We made competent cell with PCC1 vector, then transferred plasmid J61002 into a PCC1 competent cell of E.coli with PCC1 vector, using dual resistance to pick out the colony with two plasmids.

We use pCC1 vector which is a CopyControl vectors to tune gene expression. On LB chloramphenicol plates or in LB media supplemented with chloramphenicol, CopyControl clones grown in TransforMax EPI300 E. coli replicate at single-copy number from the F-factor replicon because expression of the trfA gene is repressed. Addition of CopyControl Induction Solution induces the cells to express the trfA gene product and induces their replication at high-copy number from oriV. Replication from oriV results in higher yields and higher purity of cloned DNA. The strains we use is TransforMax EP1300 Chemically competent which lack the tonA gene and are engineered for use with CopyControl Cloning Systems. The cells contain an inducible mutant trfA gene whose gene product is required for initiation of replication from the oriV origin of replication.

Fig.8 A. The device can degrade 3-PBA completely B Plasmid profile of PCC1.

When testing the degradation, we added two kinds of antibiotics (Ampicillin and Chloramphenicol ) into the medium to prevent the loss of plasmids. we added copper ion into medium(50μmol/L,final concentration of copper ion)to induce copA promoter. Our engineered bacteria can degrade about 20-60% of 3-PBA within 60 hours.(Fig.9A) We found the obvious degradation results of engineering bacteria in 200μmol/L 3-PBA solution(Fig.9B).We tested different growth situation between Sphingobium wenxiniaeJZ-1T, our engineering bacteria and CK(E.coli DH5α). The result shows that our engineering bacteria can tolerate higher concentration of 3-PBA than Sphingobium wenxiniaeJZ-1T and grow fastest in the solution. (Fig.9C)

Fig.9A Engineering bacteria`s degradation of 3-PBA . B. the result of degradation with engineering bacteria in 200μmol/L 3-PBA solution C Growth situation of different bacteria in different concentration of 3-PBA.

5.Proof of 3-HBA's induction

The device consists of regulator mhbR, mhbpt promoter and reporter .(Fig.10) We assumed that 3-HBA combined with protein mhbR was straightly contacted with the mhbpt promoter and induced the transcription process.[3] mhbpt promoter contains forward promoter and reverse promoter .They overlap with each other and contain the element necessary for induction .

Fig.10 A device consists of regulator mhbR,mhbpt promoter and reporter.

To test the induction of 3-HBA to promoter Mhbpt, we used 3-hydroxybenzoate with different concentration to induce the Mhbpt promoter, then tested the fluorescent quantity to analyze the level of induction using flow cytometer.

We found that the promoter is induced by 3-hydroxybenzoate, since the fluorescent quantity showed a significant rise with the increase of concentration of 3-hydroxybenzoate. The fluorescent quantity in solution of 5000μmol/L of 3-HBA is nearly twice of fluorescent quantity in solution without 3-HBA.(Fig.11)

Fig.11 The induction of 3-HBA to promoter Mhbpt.

References

[1] Xu Y, Gao X, Wang SH, Liu H, Williams PA, Zhou NY. MhbT Is a Specific Transporter for 3-Hydroxybenzoate Uptake by Gram-Negative Bacteria. Applied & Environmental Microbiology 2012;78:6113-20.

[2] He Z, Parales RE, Spain JC, Johnson GR. Novel organization of catechol pathway genes in the nitrobenzene degrader Comamonas sp. JS765 and its evolutionary implication. Journal of Industrial Microbiology & Biotechnology 2007;34:99-104.

[3] Lin LX, Hong L, Zhou NY. MhbR, a LysR-type regulator involved in 3-hydroxybenzoate catabolism via gentisate in Klebsiella pneumoniae M5a1. Microbiological Research 2010;165:66-74.