Difference between revisions of "Team:Jilin China/Basic Part"

wt-Pr is a constitutive promoter which is in the upstream of DmpR, a transcriptional factor of dmp operon from Pseudomonas sp. Strain CF600 encoded by dmpR gene[1].

CphA-1, a catechol 1,2-dioxygenase from Arthrobacter chlorophenolicus A6, is responsible for ring cleavage in aromatic compounds degrading process [2].

Figure 2. Reaction of cphA-1

CaO19, a hydroxyquinol 1,2-dioxygenase from Candida albicans TL3, is responsible for ring cleavage in aromatic compounds degrading process[3].

Figure 3. Reaction of CaO19

CbtA is a protein found in crytic prophage CP4-44 in Escherichia coli K-12. CbtA could inhibit cell division and cell elongation via direct and independent interactions with FtsZ and MreB[4], so it is defined as a kind of toxin.

CbeA is a protein found in crytic prophage CP4-44 in Escherichia coli K-12 which could suppress the effect of CbtA, so it is defined as a kind of antitoxin. Instead of interacting with CbtA, CbeA directly binds MreB and FtsZ and promotes the assembly of FtsZ and MreB filaments[4].

Figure 4. TA system

This year we use CbtA and CbeA to build the Geneguard system in our project. For detailed information about toxin/antitoxin (TA) system, please visit ...

DmpR is a σ54-dependent transcriptional factor of dmp operon from Pseudomonas sp. Strain CF600.[5] Transcription of Po, promoter of dmp operon, is activated when DmpR detects the presence of certain phenol compounds.[5-6] DmpR directly interacts with proper inducers at its effector-sensing domain, and the effector-sensor compound then binds to Po promoter and downstream transcription is initiated. The DmpR we use is based on the wild type DmpR used by 2013 Peking (BBa_K1031211) but has 5 sites of nucleotides mutation[7] of which two are nonsense mutation and others lead to two amino acids change. The mutant type shows high efficiency of transcription initiation.

Figure 5. The mechanism of DmpR sensor.

TfdB-JLU is a novel 2,4-dichlorophenol hydroxylase whose amino acid sequence exhibits less than 48% homology with other known TfdBs. The rate-limited step of phenolic degradation is the ortho hydroxylation[8]. Compared to wild-type TfdB, TfdB-JLU has a wilder substrate range and higher catalysis activity. Thus, the enzyme has advantages in efficient disposing of phenolic effluents[8].

Figure 6. Reaction of TfdB-JLU

[1] Shingler, V., M. Bartilson, and T. Moore. Cloning and nucleotide sequence of the gene encoding the positive regulator (DmpR) of the phenol catabolic pathway encoded by pVI150 and identification of DmpR as a member of the NtrC family of transcriptional activators. J. Bacteriol. ( 1993) 175: 1596–1604.

[2] Seok H. Lee. Effective biochemical decomposition of chlorinated aromatic hydrocarbons with a biocatalyst immobilized on a natural enzyme support. Bioresource Technology. (2013) 141:89–96.

[3] Purification and characterization of a catechol 1,2-dioxygenase from a phenol degrading Candida albicans TL3. San-Chin Tsai · Yaw-Kuen Li Arch Microbiol. (2007) 187:199–206.

[4] Masuda, Tan. YeeU enhances the bundling of cytoskeletal polymers of MreB and FtsZ, antagonizing the CbtA (YeeV) toxicity in Escherichia coli. Molecular Microbiology. (2012) 84(5), 979–989.

[5] V. L. Campos. Detection of Chlorinated Phenols in Kraft Pulp Bleaching Effluents Using DmpR Mutant Strains Bull. Environ. Contam. Toxicol. (2004) 73:666–673

[6] V. L. Campos,1 Monitoring Phenolic Compounds During Biological Treatment of Kraft Pulp Mill Effluent Using Bacterial Biosensors Bull. Environ. Contam. Toxicol. (2006) 77:383–390

[7] ARLENE A. WISE AND CHERYL R. KUSKE*. 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.

[8] Yang Lu • Ying Yu • Rui Zhou, 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