Part:BBa_K1031222

Po-B0032-sfGFP-Terminator (DmpR)

For detailed information concerning DmpR and Po promoter, please visit 2013 Peking iGEM Biosensor DmpR

Structure

Po promoter which is activated by DmpR, is σ-54 dependent. It is composed of three regions. The UAS sites containing two inverted binding region is responsible for interaction with DmpR transcriptional factor. The two IHF binding sites allowing IHF to participate, enhance transcription efficiency. -24 and -12 region interact with σ-54 factor of RNA polymerase, enabling the formation of open complex. (Fig 1)

Fig 1 Po promoter structure. The UAS of this promoter marked in green is combined of two parts in contrast direction to which DmpR binds. The box with yellow background represents IHF binding sites. The box with pink background represents σ54 binding site with -24 region and -12 region marked in red. The G with right angle represents +1 site.

DmpR

DmpR bioinformatically mined from Pseudomonas sp.CF600 ^[1-6] is a σ⁵⁴-dependent transcriptional factor that tightly controls the expression of the dmp operon (dmpKLMNOPQBCDEFGHI) (Fig. 1). This operon carries genes encoding enzymes for the degradation of (methyl) phenols into pyruvate and acetyl-CoA^[7] (Fig. 2).

Figure. 1. The schematic structure of dmp operon. Dmp operon carries genes encoding enzymes for the degradation of (methyl-)phenols to pyruvate and acetyl-CoA, the intermediates of TCA Cycle. The operon is positively controlled by the dmpR gene product, resulting in expression of catabolic enzymes when inducers like phenol are present.

Figure. 2. The catabolic pathway of phenol controlled by the dmp operon. Metabolic enzymes along the pathway are (from Step 1 to Step 8): 1, phenol hydroxylase (PH) ; 2, catechol 2, 3-dioxygenase (C23O); 3, 2-hydroxymuconic semialdehyde hydrolase (2HMSH); 4, 2-hydroxymuconic semialdehyde dehydrogenase (2HMSD) ;5, 4-oxalocrotonate isomerase (4OI); 6, 4-oxalocrotonate decarboxylase (4OD) ;7, 2-oxopent-4-cnoate hydeatase (OEH); 8, 4-hydroxy-2-2oxovalerate aldolase (HOA).

DmpR protein consists of four domains (Fig. 3): Domain A is the effector-sensing domain, which undergoes conformational change when exposed to proper inducers, including phenol, 2-chlorophenol, 2,4-dichlorophenol, methyl-phenols and other substituted phenols ^[3][8]. Domain B is a linker domain where mutations would modulate the interactions between Domain A and Domain C. Domain C is the transcriptional activation domain. Domain D contains a helix-turn-helix motif, which is responsible for the DNA binding at Po promoter ^[1].

Figure. 3. The schematic structure of DmpR protein. From N-terminal to C-terminal are Domain A, Domain B, Domain C, Domain D.

Mechanism

The mechanism of Po promoter activation consists of four steps, DmpR dimerization, DmpR hexamer formation, DNA bending and RNAP recruitment (Fig. 4). Ater the 4 steps, with the help of IHF, transcription from Po promoter initiates thereby.

Figure. 4. The mechanism of transcription activation by DmpR. (a) The inactive dimer binds to its inducer, which results in a protein conformational change. (b) Binding of ATP triggers multimerization of the dimers to hexamers (or haptamer). (c) ATP hydrolysis coupled with RNA polymerase recruitment triggers transcription activation. (D) Dissociation of the hexamers into dimers after ATP hydrolysis ^[6].

We collected and analyzed all of the information about DmpR. See Table 1 for the comprehensive summary of DmpR mutants and accompanied novel aromatics-sensing characteristics, which provides a rich repertoire for the synthetic biologists to customize the aromatics-sensing characteristics of DmpR protein.

Sequence and Features

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 211
  

Construction and data

Peking iGEM has adopted DmpR to build a biosensor circuit (Fig. 6). Plasmid carrying Pr-DmpR was co-transformed with the plasmid containing the inducible promoter Po and reporter gene sfGFP (Fig. 6). Similar to other biosensors, plasmid with RBS BBa_B0032 preceding sfGFP was chosen due to its relatively higher induction ratio during primary test for the RBS library.