Team:Jilin China/Composite Part
The function of the composite part we have been working on is to detect and degrade chlorophenol under population control of TA system.
Figure 1: The TA - DmpR - TfdB-JLU construct
This part is a combination of our Geneguard system and chlorophenol response system. Part BBa_J23107 is a constitutive promoter which could provide a constitutive expression of sensor DmpR and toxin CbtA.
DmpR is an σ54-dependent transcriptional factor of dmp operon from Pseudomonas sp. Strain CF600.[1] Transcription of Pdmp, promoter of dmp operon, is activated when DmpR detects the presence of certain phenol compounds.[1-2] DmpR directly interacts with proper inducers at its effector-sensing domain, and the effector-sensor compound then binds to Pdmp 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[3](Shows below). The mutant type shows high efficiency of transcription initiation.
Figure 2. Mutant type DmpR and wild type DmpR
Toxin CbtA could keep the growth of the engineered bacterium in a low level. When exposed to contaminants, complex of DmpR and chlorophenol could bind to promoter Po and initiate the expression of antitoxin CbeA as well as TfdB-JLU, a monooxygenase that is responsible for initial hydroxylation of the benzene ring[4-5].
Figure 3. Reaction of TfdB-JLU
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,1 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 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.
[4] Ledger T, Pieper DH, Gonzalez B. (2006) Chlorophenol hydroxylases encoded by plasmid pJP4 differentially contribute to chlorophenoxyacetic acid degradation. Appl Environ Microbiol 72:2783–2792.
[5] Yang Lu. (2011) Cloning and characterisation of a novel 2,4-dichlorophenol hydroxylase from a metagenomic library derived from polychlorinated biphenyl-contaminated soil. Biotechnol Lett. 33:1159–1167
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