Difference between revisions of "Part:BBa K1031222"

Latest revision as of 16:04, 27 October 2020

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

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 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 presence of an inducer of biosensor A (not an inducer of biosensor B) doesn’t interfere with the dose response of biosensor B to any of its inducers, and vice versa, we call the B and A biosensors are "orthogonal"; namely, no synergistic/antagonistic effects happen between the inducers of A and B biosensors.(for more details, Chick Here)

Sensor	Host	Main Inducers
XylS	Pseudomonas putida	BzO 2-MeBzO 3-MeBzO 2,3-MeBzO 3,4-MeBzO
NahR	Pseudomonas putida	4-MeSaA 4-C1SaA 5-C1SaA SaA Aspirin
DmpR	Pseudomonas sp.600	Phl 2-MePhl 3-MePhl 4-MePhl 2-ClPhl
HbpR	Pseudomonas azelaica	o-Phenylphenol 2,6'-DiHydroxybiphenol

Figure.8 Summary of the orthogonality assay to evaluate the synergistic/antagonistic effects between the inducers of 4 representative biosensors.
No synergistic or antagonistic effects between the inducers of 4 representative biosensors (XylS, NahR, HbpR, and DmpR) were observed. For instance, although the sensing profiles of NahR and XylS overlap to some extent, the NahR-specific and XylS-specific inducers proved to be really orthogonal.

We have confirmed the orthogonality among inducers of different biosensors, which is one of the main features we expect for our aromatics-sensing toolkit; this allowed the combination of these biosensors to profile aromatics for the ease of practical applications.

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

We examined the orthogonality between 4 representative biosensors (Fig.9). The orthogonality test between two biosensors, biosensor I and biosensor II, was performed in the following procedure:

1. A typical inducer A for biosensor I and a typical inducer B for biosensor II were selected.
2. The dose response of biosensor I to inducer A was measured, under the perturbation of inducer B.
3. The dose-response of biosensor II to inducer B was measured, under the perturbation of inducer A.

If biosensor I and biosensor II are orthogonal, the dose response of biosensor I to inducer A should be constant, regardless of the concentrations of inducer B; and the dose response of biosensor II to inducer B should be constant, regardless of the concentrations of inducer A. Namely, for two "orthogonal" biosensors, the perturbation of an unrelated inducer has negligible effect on the dose response of a biosensor to its related inducer (Fig.10).

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.

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 orthogonality between XylS, NahR, HbpR and DmpR biosensors have been carefully evaluated using the assay discussed above (Fig.9). The data were processed by linear fitting and the slopes of the fitting curves were compared with 1 (Fig.9, Fig.10). The closer the slope was to 1, the more orthogonal the two biosensors were. Results showed that the biosensor pairs, XylS and NahR (Fig.11a, b), XylS and HbpR (Fig.11c, 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 orthogonal, as summarized in Fig.8.

Figure.11 Linear fitting of the data obtained from the orthogonality assay showing that the orthogonality between the 4 representative biosensors. The experiments and data processing were performed as described in Fig.9 and Fig.10.The black dashed line denotes slope=1 as the reference line. These fittings showed the orthogonality between 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 experiment data, linear fitting curves of biosensor, and cognate inducers are in different colors: XylS in red, NahR in green, HbpR in orange and DmpR in dark cyan.

TPR_China iGEM2020

This part is not such good in our own experiment.

To characterize the property of the device itself, the original aromatic compond o-Cresol (oCl) was used as inducer. According to the experment results provided by Peking iGEM2013, the oCl could cause the largest dynamic range and the most sensity of the DmpR sensor.

The fluorescence of the sensor induced by small molecules

Through the corresponding small molecule sensing experiment, except Dmpr sensor, all our sensors have great induction effects on its corresponding aromatic small molecules. Among which NahR-sfGFP was the best! It is 63 times of the negative control.And here we can see that Xyls-sfGFP is 18 times of the Xyls-GFP( BBa_K1031911). And what surprises us is that Paax sensor also has a good response effect to small molecules, which has not been detected before.

The fluorescence of the sensor induced by small molecules

The fluorescence of the NahR sensor induced by small molecules

The fluorescence of the sensor induced by PAN

And here is the fluorescence of different sensors induced by PAN.This data shows the same result that NahR-sfGFP responded best.

The fluorescence of the sensor induced by PAN

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.

@@ Line 224: / Line 224: @@
 This part is not such good in our own experiment.
+To characterize the property of the device itself, the original aromatic compond o-Cresol (oCl) was used as inducer. According to the experment results provided by Peking iGEM2013, the oCl could cause the largest dynamic range and the most sensity of the DmpR sensor.
 ====The fluorescence of the sensor induced by small molecules====