Difference between revisions of "Part:BBa K1031222"

Revision as of 16:28, 27 September 2013

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).

Sequence and Features

Sequence and Features

Construction and data

We constructed a library of RBSs (Ribosome Binding Sites) of different strength for tuning the expression of reporter sfGFP. K1031222 is a reporter circuit composed of three elements, the inducible promoter Pofor DmpR, RBS B0032[1], and reporter gene sfGFP. (Fig 2)

Fig 2 Construction of reporter circuit. The orange arrow represents Po promoter for DmpR. The green oval stands for RBS B0032. sfGFP coding sequence is shown with dark blue, while terminator B0015[2] is in dark red.

The performance of reporter circuits adopting B0031 and B0032 in collocation with Pc-DmpR were tested.(Fig 3) The dashed box refers to data for Po/DmpR biosensor circuit adopting RBS B0032. (Fig 3)

Fig 3 Fluorescence intensity of DmpR biosensor at the absence and presence of inducer. The line of light blue represents fluorescence background, and the line of dark blue stands for fluorescence intensity when exposed to phenol. The dashed box refers to data for reporter circuit adopting B0032. From data it is obviously the circuit adopting B0032 is superior.