Difference between revisions of "Part:BBa K1031222"

Revision as of 01:46, 28 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)

Figure.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 is a σ⁵⁴-dependent transcriptional factor that tightly controls the expression of the dmp operon (dmpKLMNOPQBCDEFGHI) from Pseudomonas sp.CF600 ^[1-6](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

Construction and Data

Peking iGEM has adopted DmpR to build a biosensor circuit contain Po promoter(Fig. 5). Plasmid carrying Pr-DmpR was co-transformed with the plasmid containing the inducible promoter Po and reporter gene sfGFP (Fig. 5). 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.

Figure.5 The schematic diagram for the DmpR biosensor circuit.
DmpR is constitutively expressed and functions to regulate the transcription of sfGFP gene via promoter Po. As for the RBS of sfGFP, BBa_B0032 was decided due to its better performance compared to RBS of other translation strengths.

We evaluated the performance of DmpR using our own protocols and almost every protocol mentioned in the previous studies (for more details about these three protocols, Test Protocol 1-3, Click Here). Results showed that the Test Protocol 3 works the best, with which we tested the ON-OFF ratios of all the 78 aromatic compounds. 7 compounds out of the 78 showed significant induction ratios (>2) (Fig. 7): Phl, 2-MePhl, 2-ClPhl, 3-ClPhl, Cat, 4-NtPhl and 2-APhl (Click Here for the full names of the aromatic compounds). The presence of phenol is consistent with previous studies. The other 6 compounds, however, have not been reported according to our knowledge.

Figure.6 ON/OFF test to evaluate the induction ratios of all aromatic compounds in the aromatics spectrum (For the full names of the compounds, Click Here).
(a) The induction ratios of all 78 aromatic species for the DmpR biosensor. The DmpR biosensor could respond to 7 out of the 78 aromatics with the induction ratio higher than 2. (b) The aromatics-sensing profile of DmpR biosensor. The aromatic species that can elicit strong responses of DmpR biosensor are highlighted in cyan in the aromatics spectrum. The structure formula of typical inducer is also presented around the spectrum. The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to object inducers by the basal fluorescence intensity of the biosensor itself.

To provide more detailed information about the aromatics-sensing profile, we carefully examined the individual dose-response curves of the 7 compounds via Test Protocol 3

Figure.7 Dose-response curves of DmpR biosensor induced by 7 strong inducers (phenol, its homologs and derivatives). The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to inducers by the basal fluorescence intensity of the biosensor. For the full names of the compounds, Click Here .

In summary, we have successfully constructed the DmpR biosensing circuit and fine-tuned it guided by our experience obtained from the building of other biosensors. The aromatics-sensing profile of DmpR biosensor is considerably narrow (Fig. 7), making it a robust and convenient biosensor for the presence of phenol and its derivatives.

Orthogonality of Different Sensor

If the existence of each inducer A, which is not an inducer of B, doesn’t interfere the B’s dose-response to any of its inducers, vice versa, we call the biosensor A and B are orthogonal, and we are secured to apply the multi-component analysis with A and B.(for more details, Chick Here)

Figure.8 Summary of the orthogonality among four sensors.

we have confirmed the orthogonality among inducers of different biosensors, which is one of the main features we expect for our aromatics-sensing toolkit. Our sensors are well suited to multi-component analysis.

Related Parts:

XylS: https://parts.igem.org/Part:BBa_K1031911 Wiki: http://2013.igem.org/Team:Peking/Project/BioSensors/XylS

NahR: https://parts.igem.org/Part:BBa_K1031610 Wiki: http://2013.igem.org/Team:Peking/Project/BioSensors/NahR

HbpR: https://parts.igem.org/Part:BBa_K1031300 Wiki: http://2013.igem.org/Team:Peking/Project/BioSensors/HbpR

DmpR: http://2013.igem.org/Team:Peking/Project/BioSensors/DmpR

Orthoganaility between inducer A (originally detected by biosensor I) and B (originally detected by biosensor II) were tested in the following manner (Fig.9). To test the effect of inducer B upon the dose-response curve of inducer A obtained by biosensor I:

(1) Fluorescence intensity of biosensor I elicited by inducer A of concentration gradient was measured as standard results (Fig.9a, Lane 1);

(2) And fluorescence intensity of biosensor I induced by inducer A of concentration gradient in the presence of a certain concentration of inducer B was measured (Fig.9a, Lane 2 and 3) and compared with the standard results.

The effect of inducer A upon the dose-response curve of inducer B obtained by biosensor II was tested vice versa (Fig.9b).

Figure.9 Orthogonality test assay for inducer A (detected by biosensor I) and inducer B (detected by biosensor II). (a) Biosensor I was added into the test assay. Different mixtures of inducers were added into lane 1, 2, and 3 respectively as listed above. Effect of inducer B upon the dose-response curve of inducer A was tested by comparing the fluorescence intensity of biosensor I among lane 1 ,2, and 3. (b) Biosensor II was added into the test assay. Different mixtures of inducers were added into lane 1, 2, and 3 respectively as listed above. Effect of inducer A upon the dose-response curve of inducer B was tested by comparing the fluorescence intensity of biosensor II among lane 1 ,2, and 3.

If biosensor I and biosensor II are orthogonal, the dose-response of biosensor I is invariant to the concentration of inducer B and the dose-response of biosensor II is invariant to the concentration of inducer A. This invariance is showed in the form of Fig.10.

Figure.10 Correlation of the inducer B and the dose-response of biosensor I to its inducer A. Each point on the right plot represents a concentration of inducer A. It's x coordinate represents the fluorescence when inducer B is 0 and the y coordinate represents the fluorescence when the cell is exposed to a none-zero concentration of inducer B. If the dose-response of biosensor I is invariant to the concentration of inducer B, the x coordinate of a experimental point should be equal to its y coordinate and the experimental points are supposed to be aligned in a line whose slope is one.

The orithogonality of XylS, NahR, HbpR and DmpR biosensors have been carefully confirmed using the test assay introduced above (Fig.11). The experimental points were processed by linear fitting and the slopes of the fitting curves were compared with 1. The closer the slope was to 1, the more orthogonal the biosensors were. The results showed that biosensor XylS and NahR (Fig.11a,b), XylS and HbpR (Fig.9c,d), NahR and HbpR (Fig.11e,f), XylS and DmpR (Fig.11g,h), NahR and DmpR (Fig.11i,j), and HbpR and DmpR (Fig.11k,l) are all investigated to be orthogonal, which is summarized in Fig.8

Figure.11 Experimental points and the linear fitting curves of the orthogonality test. The black dashed lines are with the slopes of 1, showing as the reference line. The slopes of the experimental fitting curves were showed in the upside portion of the figure, all of them were around 1. These data showed the orthogonality among biosensors(a, b) XylS and NahR; (c, d) XylS and HbpR; (e, f) NahR and HbpR, (g, h) XylS and DmpR, (i, j) NahR and DmpR, and (k, l) HbpR and DmpR. The experimental points and linear fitting curves of biosensor and its inducers are marked in different colors: XylS in red, NahR in green, HbpR in orange and DmpR in dark cyan.

Reference

[1]. SHINGLER, V.; PAVEL, H. Direct regulation of the ATPase activity of the transcriptional activator DmpR by aromatic compounds. Molecular microbiology, (1995), 17.3: 505-513.
[2]. SHINGLER, Victoria; MOORE, Terry. Sensing of aromatic compounds by the DmpR transcriptional activator of phenol-catabolizing Pseudomonas sp. strain CF600. Journal of bacteriology, (1994), 176.6: 1555-1560.
[3]. SZE, Chun Chau; LAURIE, Andrew D.; SHINGLER, Victoria. In Vivo and In Vitro Effects of Integration Host Factor at the DmpR-Regulated σ⁵⁴-Dependent Po Promoter. Journal of bacteriology, (2001), 183.9: 2842-2851.
[4]. SARAND, Inga, et al. Role of the DmpR-mediated regulatory circuit in bacterial biodegradation properties in methylphenol-amended soils. Applied and environmental microbiology, (2001), 67.1: 162-171.
[5]．WISE, Arlene A.; KUSKE, Cheryl R. Generation of novel bacterial regulatory proteins that detect priority pollutant phenols. Applied and environmental microbiology, (2000), 66.1: 163-169.
[6]. TROPEL, David; VAN DER MEER, Jan Roelof. Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiology and Molecular Biology Reviews, (2004), 68.3: 474-500.
[7]. SHINGLER, V.; POWLOWSKI, J.; MARKLUND, U. Nucleotide sequence and functional analysis of the complete phenol/3, 4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600. Journal of bacteriology, (1992), 174.3: 711-724.
[8]. GUPTA, Saurabh, et al. An Effective Strategy for a Whole-Cell Biosensor Based on Putative Effector Interaction Site of the Regulatory DmpR Protein. PloS one, (2012), 7.8: e43527.