This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_J61051

The following data is from Peking iGEM 2013 team, uploaded by ZHENG Pu.

Construction and Test

Construction

We placed the Standard Biological Part BBa_J61051 which contains the constitutively expressed NahR and sal promoter in front of the reporter gene sfGFP (Fig.5) via standard assembly. The plasmid verified by Beijing Genomics Institute was transformed into E. coli (TOP10, TransGen Biotech). Single clone of bacteria was picked and grown in rich LB medium added chloromycetin (170 μg/ml) overnight and stored at -80℃ in 20% glycerol, waiting for induction test.

Fig.5. Schematic diagram for the NahR biosensor circuit. The Standard Biologicla Part BBa_J61051 was placed preceding reporter sfGFP in the backbone pSB1C3. Promoters are presented in orange, RBS in light green, coding sequence in dark blue and terminators in red.

On-Off Tests

Fig.6. Response of sensor NahR biosensor to various aromatic species. (For the full name of the compounds, CLICK HERE). (a) The induction ratio of various aromatic species in the ON-OFF test. NahR could respond to 18 out of 78 aromatics with the induction ratio over 20. (b) The aromatics-sensing profile of NahR biosensor.The aromatic species that can elicit strong responses of NahR biosensor are highlighted in green in the aromatics spectrum. The structure formula of typical inducer is also listed around the spectrum. Induction ratio is calculated by dividing the fluorescence intensity of biosensor exposed to inducers by the basal fluorescence intensity of the biosensor.

Dose-response Curve

Fig.7 Dose response curves of NahR biosensor. (a) Dose response curves for salicylate, its homologs and derivatives; (b) Dose response curves for benzoate, its derivatives and special inducers like 5-ClSaD and 2,4,6-TClPhl. Induction ratio is calculated by dividing the fluorescence intensity of biosensor exposed to inducers by the basal fluorescence intensity of the biosensor.For the full name of the compounds, CLICK HERE).

Orthogonality

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

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 multicomponent 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.1). 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.1a, 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.1a, 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.1b).

Fig.1. 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.

We managed to demonstrate the orthogonality among inducers of different biosensors in a more quantitative and visible way. If inducer A and B were orthogonal, the fluorescence intensity should be identical no matter with or without the irrelevant inducer B. That is to say, the ideal experimental points should be aligned in a line whose slope is one.

The orithogonality of inducers of XylS, NahR, HbpR and DmpR biosensors have been carefully confirmed using the test assay introduced above (Fig.2). 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 inducers were. The results showed that inducers of biosensor XylS and NahR (Fig.2a, b), XylS and HbpR (Fig.2c, d), NahR and HbpR (Fig.2e, f), XylS and DmpR (Fig.2g, h), NahR and DmpR (Fig.2i, j), and HbpR and DmpR (Fig.2k, l) are all highly orthogonal, which is summarized in Fig.2.

Fig.2. 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 inducers of 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.

User reviews:

Uppsala iGEM 2015 Review

The Uppsala igem team characterize this part as an inducible promoter with the RFP dTomato as a reporter.

Figure 14. dTomato expressed in E. coli DH5-alpha. NahR/Psal induction test in LB medium. Concentration of salicylate from left to right: 0,1 μM, 1 μM, 10 μM, 100 μM and 1 mM.

Figure 15. dTomato expressed in E. coli DH5-alpha. NahR/Psal induction test on agar plate. Concentration of salicylate from left to right: 0,1 μM, 1 μM, 10 μM, 100 μM and 1 mM.

We tested the NahR/Psal promoter system with dTomato on a pSB1C3 plasmid. The cells containing the plasmid were streaked on agar plates with different salicylate concentrations in the agar medium. Similarly, we also examined liquid cultures with different concentrations of salicylate in the solution. Both of these tests showed that the bacteria were producing the dTomato protein in presence of salicylate, independent of the salicylate being in liquid culture or in the solid growth medium. It can also be easily observed that the fluorescence increased with increasing salicylate concentrations (See figure 14 and 15). The measurements were made with the fluorometer that our team built in order to be able to characterize this system. These results were compared to MACS measurements on the same colonies and the measurements were correlated.

The usage of the NahR/Psal promoter system is very central in our project. Overexpressing enzymes is very energetically expensive for the cells. Therefore, regulated expression is often preferred. A previous iGEM team, Peking 2013, inspired us to use the NahR construct (BBa_J61051). The copresence of naphthalene with other higher molecular weight PAHs in industrial environments lets us take advantage of the regulatory function of one of naphthalenes metabolites, namely salicylate.

The initiation of the degradative phase of our system starts with the enzymatic breakdown of naphthalene to salicylate. Salicylate induces transcription downstream of the NahR/Psal promoter complex..

</>p>

NYMU-Taipei Review

The inducible promoter was induced with different concentration(0, 1.50E-07, 6.10E-07, 2.40E-06, 9.80E-06, 3.90E-05, 1.60E-04, 6.20E-04, 1.00E-03) every 30 minutes. We started the induction when the OD600 was 0.6-0.7. The fluorescence response was measured with plate reader.

<img src="">
The inducible promoter was induced with different concentration(0, 1.50E-07, 6.10E-07, 2.40E-06, 9.80E-06, 3.90E-05, 1.60E-04, 6.20E-04, 1.00E-03) every 30 minutes. We started the induction when the OD600 was 0.6-0.7. The fluorescence response was measured with plate reader. The red line indicate the Hill equation(P = Pmax*[SA^n]/[SA^n+Km^n] ) fit of our dose-response data with error bar showing the standard deviation and the R square value of this fit is 0.99531. "n" indicates the hill coefficient(unitless), "Pmax" indicates the maximal promoter activity, and "K" is the salicylic acid concentration while P equals 1/2*Pmax

<regulartext1>The NYMU-Taipei igem team characterize this part as an inducible promoter with GFP response as reporter. The following data was obtained by microplate reader:

<img src="">
|}

Reference

[1] Dunn, N. W., and I. C. Gunsalus.(1973) Transmissible plasmid encoding early enzymes of naphthalene oxidation in Pseudomonas putida. J. Bacteriol. 114:974-979
[2] M. A. Schell.(1983) Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7. J. Bacteriol. 153(2):822
[3] M. A. Schell, and P. E. Wender.(1986) Identification of the nahR gene product and nucleotide sequences required for its activation of the sal operon. J. Bacteriol. 116(1):9
[4] Woojun Park, Che Ok Jeon, Eugene L. Madsen.(2002) Interaction of NahR, a LysR-type transcriptional regulator, with the K subunit of RNA polymerase in the naphthalene degrading bacterium, Pseudomonas putida NCIB 9816-4. FEMS Microbiology Letters. 213:159-165
[5] Mark A. Schell, Pamela H. Brown, and Satanaryana Raju.(1990) Use of Saturation Mutagenesis to Localize Probable Functional domains in the NahR protein, a LysR-type Transcription Activator. The Journal of Biological Chemistry. 265(7): 3384-3850.
[6] Angel Cebolla, Carolina Sousa, and Vı´ctor de Lorenzo.(1997) Effector Specificity Mutants of the Transcriptional Activator NahR of Naphthalene Degrading Pseudomonas Define Protein Sites Involved in Binding of Aromatic Inducers. The Journal of Biological Chemistry. 272(7):3986-3992
[7] M. A. Schell, and E. F. Poser.(1989) Demonstration, characterization, and mutational analysis of NahR protein binding to nah and sal promoters. J. Bacteriol. 171(2):837
[8] Jianzhong Huang and Mark A. Schell.(1991) In vivo interaction of the NahR Transcriptional Activator with its target sequences. The Journal of Biological Chemistry. 266(17):10830-10838
[9] Hoo Hwi Park, Hae Yong Lee, Woon Ki Lim, Hae Ja Shin. (2005) NahR: Effects of replacements at Asn 169 and Arg 248 on promoter binding and inducer recognition. Archives of Biochemistry and Biophysics. 434:67-74