Latest revision as of 15:27, 21 October 2019

Nitrate reporter: PyeaR - GFP composite

Nitrate and Nitrite sensitive promoter PyeaR with a GFP coding device and strong RBS to create a nitrate-sensitive system which signals through expression of GFP.

PyeaR is normally repressed by NsrR, a protein native to most 'E. coli' cells. When Nitrate or Nitrite enter the cell it is converted to Nitric Oxide. This binds to NsrR, halting the repression and allowing the production of GFP.

Optimum use appears to be when sensing nitrate within a range of 0 - 20 mM (Calculated from Miller assays)

Activity between 0 - 10mM was investigated using an assay measuring change in GFP expression with increasing levels of Potassium Nitrate, the results of which can be seen below.

Additional characterization. Strain was NEB Turbo.

HFLS_H2Z_hangzhou 2017

Improve the Characterization of BBa_K381001

Team HFLS_H2Z_hanzhou's experience with this part. In our experience with this part, we found out that Pyear will yield strong transcription rate under the presence of high concentration of Nitrate. However, we found out, under high concentration of nitrite(40mM), the promoter will be repressed, unlike nitrite in low concentration. Furthermore, if nitrate(40mM) is present along with nitrite(40mM), the promoter will be inhibited.

NCKU_Tainan 2017

Improve the Characterization of BBa_K381001

BBa_K381001 was first designed by Katharine Coyte from team BCCS-Bristol in iGEM 2010. It is a nitrate reporter, PyeaR-GFP composite. The team BCCS-Bristol only test the BioBrick’s sensitivity at a higher concentration, but the nitrate level of aquatic water usually won’t reach up to 1 mM. Therefore, we carry out experiment by testing the fluorescence intensity with ppm scale of Potassium Nitrate, which is much more lower than all the teams before. The results are shown below.

It shows that the BioBrick BBa_K381001 have nice sensitivity not only in high nitrate concentration, but low concentration as well. Many teams had done improvements of K381001, but our team bring out the most beautiful data. And we also take X axis logarithm, which can make our data linear. Not only easier to read, but also more meaningful. The data is shown as below.

Improve the Function of BBa_K381001

PyeaR is repressed by NsrR protein under no nitrate or nitric oxide condition, and is activated when nitrate or nitrite is existing.

In theory, the biobrick K381001 can’t emit fluorescence under no nitrate or nitrite. However, the data showed that it can still be activated. In order to improve the biobrick K381001 and decrease the fluorescence basal level, we decided to add an additional nsrR sequence to it so as to reinforce repression and decrease interference. As a result, fluorescence basal level can be decreased, and detection will be enhanced.

To see more details about the construction and result, click the hyperlink below:

>> PyeaR-NsrR binding site-B0030-GFP composite : BBa_K2275011

>> PyeaR-B0030-NsrR binding site-GFP composite : BBa_K2275012

>> PyeaR-NsrR binding site-amplifier-rbs composite constructed by team HFLS_H2Z_hangzhou: [1]

NEU_China_A 2018 Improvement

The promoter PyeaR is sensitive to nitrate and nitrite. When nitrate and nitrite enter E. coli, they are converted to nitric oxide. Nitric oxide binds to the repressor protein NsrR, which inactivates PyeaR to inhibit transcription of downstream genes. Then the promoter PyeaR will be activated.

1. Usage and Biology

We learnt that iGEM 2010 Team BCCS-Bristol had used BBa_K381001 to detect the soil nitrate and nitrite to demonstrate the fertility of soil. Thus, farmers can determine which soils are fertilized by detecting the fluorescence of GFP reporter gene. In this way, farmers only need to apply fertilizer in places where there is no fertility, which can save excess fertilizer. Given the economic costs and the impact of eutrophication on ecosystems, the use of BBa_K381001 has great benefits for both agriculture and the environment. However, due to the influence of outdoor temperature, GFP fluorescence density was fluctuated significantly. This instability is unfavorable for the detection of soil fertility. In addition, the detection of GFP fluorescence signal requires special equipment that is not readily available for farmers. Therefore, we replaced GFP with blue chromoprotein (amilCP encoded protein) for visual detection. On the one hand, amilCP expression is less affected by temperature and is a more stable reporter than GFP. On the other hand, blue chromoprotein can be visualized by human eyes, instead of requiring the special equipment. Therefore, we believe that our improved part BBa_K2817007 is very beneficial to farmers.

2. Characterization

According to the results of the ShanghaiTechChina_B 2016 team, 100μM Sodium Nitroprusside Dihydrate (SNP) aqueous solution can continually release NO, and the NO concentration is stable at about 5.5μM. Since our project also tested for inflammatory signals, we chose this concentration before testing for BBa_K381001 and BBa_K2817007.

The construction of BBa_K381001 can be seen from Figure 1A. We transformed the plasmid containing BBa_K381001 into DH5α competent E. coli strain and cultured at 37 ℃ overnight to dilute to OD600 = 0.4. Then we took half of bacteria as control and the rest was added SNP aqueous solution, and induced at 37 ℃ for 6 h. Then the fluorescence intensity of cells was observed under microplate reader (Figure 1B) and fluorescence inverted microscope (Figure 1C). The histogram of GFP fluorescent density and microscope images indicated that PyeaR could effectively activated by NO and there was almost no leakage expression.

Figure 1. The test of BBa_K381001. A, the construction of BBa_K381001. B, Histogram of GFP fluorescence: LB control, without SNP, with 100μM SNP. C, GFP fluorescence image from top to bottom: without SNP, with 100μM SNP.

The construction of BBa_K2817007 can be seen from Figure 2A. We transformed the plasmid containing BBa_K2817007 into DH5α, and cultured at 37 ℃ overnight to dilute to OD = 0.4. Then we took half as control and the other half added SNP aqueous solution and induced at 37 ℃ for 6 h. We also set up negative control group which doesn’t contain amilCP. After 6 h at 37 ℃, 1 mL of the bacterial solution was centrifuged at 8,000 r.p.m for 1 min (Figure 2B). We could directly observe the result of PyeaR being activated by NO without special equipment.

Figure 2. The test of BBa_K2817007. A, the construction of BBa_K2817007. B, Pellets of bacteria transformed with plasmid containing BBa_K2817007 after induction of 6h. From left to right: negative control group, without SNP group, with 100μM SNP group.

3. Conclusion

In conclusion, we confirmed our improvement through an experimental comparison between the two parts. In the real world, our improved part BBa_K2817007 has better usability. In the future, we will further confirm the situation of different concentrations of NO and different temperature conditions.

NEU_China 2019

The improvement of BBa_K381001

This year, we chose BBa_K2967017 (PyeaR-Luc) https://parts.igem.org/Part:BBa_K2967017

as an alternative to our inflammatory sensor, due to its sensitivity to nitrate and nitrite. When nitrate and nitrite enter E. coli, they will be converted to nitric oxide. Then nitric oxide will bind to the repressor protein NsrR that inactivates PyeaR to inhibit transcription of downstream genes.[1]

However, we noticed detectable basal expression (leakage) from the characterization of the most sensitive NO sensor (PyeaR-Luc) (Fig. 2A). To reduce sensor basal background, we integrated two different approaches. For the first approach, we inserted an extra NsrR binding sequence (NsrRBS) downstream of PyeaR to create a ‘roadblocking’ effect [2] (Fig. 1). Compare to the unmodified Pyear-luc system (Fig.2B), the histogram of luminescence data demonstrated that the relative lower luciferase signal in Pyear-NsrRBS system in the absence of NO.

Figure 1. Diagram for NO sensor system in pCDFDuet-1 plasmid. PyeaR, a promoter which is sensitive to NO. Native NsrRBS, the native NsrR binding sequence. Extra NsrRBS, the extra NsrR binding sequence. Luciferase, reporter gene.

Figure 2. The response to NO sensors. A. The response to NO of Pyear-luc in ECN. Histogram of Luminescence(RLU): pcdfduet-1 blank, Pyear-luc without SNP, pcdfduet-1 blank, Pyear-luc with 100μM SNP. B. Comparison genetic leakage expression of Pyear-luc and Pyear-NsrRBS-luc systems with or without NO induction. Blue bars indicate the luciferase expression percent under the NO induction, while Red bars show the percentage of genetic leakage without NO induction.

The second approach uses protease-based post-translational degradation regulation[2]. First a protein degradation tag (AAV) is added to the reporter protein to reduce the output basal expression. To reduce the background expression without sacrificing the high output, we next incorporated the sensor into a TEV protease-based reporter protein degradation control system (Fig. 3). This hybrid regulation system is sufficient to reduce the sensor’s basal background while also being able to maintain both the sensor’s output amplitude and sensitivity, leading to expanded output dynamic range. However, due to the time limitation, the result is not shown here.

Fig. 3 Tuning the sensor background and output dynamic range via reporter degradation regulation. Schematic showing protease-mediated regulation of the background and output dynamic range for an NO sensor. ‘A’ represents the AAV degradation tag. Off state: when there is no NO induction. On state: when there is NO induction.

reference

[1] Lin, H. Y., Bledsoe, P. J., & Stewart, V. (2007). Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen-responsive regulator Fnr in Escherichia coli K-12. Journal of bacteriology, 189(21), 7539-7548.

TAU_Israel 2019

The Characterization of BBa_K381001

We decided to characterize the Nitrate reporter, PyeaR - GFP composite (BBa_k381001). This part was originally created by BCCS-Bristol in iGEM 2010, and it was further improved and characterized by other groups. However, these characterizations did not qualify for the current measurement standard as they did not integrate the MEFL/Particles standardized units. Therefore, we further characterized this part by measuring its expression levels in E. coli DH10β using standardized fluorescein/Beads units, following the iGEM measurement kit.

Characterizations Outline:

1. Preform iGEM's calibration protocols according to the Measurement Hub:
   1. Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles V.2, according to the protocol published here - [2]
   2. Calibration Protocol - Plate Reader Fluorescence Calibration V.3 , according to the protocol published here - [3]
2. Transform DNA from the iGEM distribution kit 2019 into E. coli DH10 β.
3. Compare fluorescence levels of overnight-grown E. coli cultures.
4. Measure OD₆₀₀ and GFP fluorescence levels of the inducible E. coli samples over time with increasing concentrations of the NO3 inducer, and subtract from them the mean reading obtained in the blank samples (media only).
5. Compare fluorescence levels of overnight incubated BioBricks starters.
6. Measure GFP fluorescence levels of the inducible BioBrick (BBa_k381001) and compare it to the fluorescence level of both the LB\CM medium and the Constitutive GFP BioBrick (BBa_K608011).
7. Transform the data from Arbitrary fluorescence and OD₆₀₀ units into MEFL and Particles units, respectively, using the iGEM Measurement hub excel charts (Here).
8. Calculate the MEFL/Particle ratio, and display our results with respect to these units

Measurement Protocol:

1. Incubate bacteria overnight in 3ml LB at 37 ℃
2. Prepare inducer working stock of 1 M by dissolving NaNO3 in ultra-pure water.
3. Prepare inducer dilution stocks in LB/CM.
4. Bacteria preperation
   1. Transfer 1 ml of overnight-grown bacterial culture into a new 1.5 ml eppendorf tube.
   2. Measure OD₆₀₀ in a spectrophotometer.
   3. Pellet culture volume containing 1 OD units by centrifugation for 1 minute at 16100 g at room temperature, and remove the supernatant
   4. Resuspend bacteria pellet by adding 1 ml LB followed by vortexing. This is the 1 OD₆₀₀ working stock
5. Prepare the 96 well plate as described in Figure 1 (or possibly any other structure) by adding both bacterial samples and adequate Nitrate solution quantity.
6. Enter the 96 well plate into the microplate reader, and measure both OD₆₀₀ and GFP fluorescence (we used gain of 50; excitation at 485 nm; emission at 528 nm in a 'BioTek Synergy H1 reader').

'Figure 1. Plate layout used in the analysis.. Measurement of both the optical density OD₆₀₀ and GFP fluorescence was done using a 'BioTek Synergy H1' plate reader. Turbidity of bacterial suspensions was determined at 600 nm, while for GFP fluorescence we used a gain of 50, and excitation at 485 nm / emission at 528 nm.

Results

In general, the Bio-Brick worked as expected; the introduction of nitrate did induce the GFP production, and thus the fluorescence levels. It appears that higher inducer concentrations (above 10 μM) did not result in an equivalent effect on the MEFL/particle ratio. A possible explanation could be that inducer concentration above 10 μM saturated the expression from the promoter. Another possible explanation can be that high concentrations of the inducer may cause a nutrient problem for the bacteria in the growth medium. These measurement results demonstrate that a nitrate concentration of about 10 μM generated the optimal fluorescence level, in agreement with previously reported results. In addition, we now describe the BioBrick's behavior by using standardized units of MEFL and number of particles.

Figure 2. Change in the MEFL/Particles ratio (Molecules of Equivalent Fluorescein divided by the number of Particles) as a function of time (Minutes). Conversion ratio from Fluorescence arbitrary units to MEFL, and of Abs600 to Particles was done using the tools supplied by the iGEM 2019 Measurement hub.

IISER Kolkata 2019

The Characterization of BBa_K381001

Our model relies on the ability of the bacterial chassis to detect the various Nitric Oxide (NO) concentrations inside the macrophage and trigger the modified genetic circuit accordingly. Thus as proof of model we are characterizing the induction of PyeaR promoter in different concentrations. For experimental purposes, we are using Sodium Nitroprusside (SNP) as the NO donor. From literature, it is known that one molecule of SNP releases one molecule of NO in aqueous solution.

Aim

BBa_ was used as a test circuit to quantify the PyeaR promoter activity in different concentrations of NO in the LB medium.

Method

E. coli DH5α were transformed using re-suspended BBa_k381001 in pSB1C3 plasmids from iGEM 2019 distribution kit and selected using Chloramphenicol LB agar plates.

1. Single positive colony of BBa_k381001 was added to 10ml of LB media and left overnight at 37° C on 150 rpm in an BOD incubator.
2. 4 mL of overnight culture was added to 200 mL of LB media and incubated until the OD reached 0.4.
3. The secondary culture was divided into five flasks each with 50mL and calculated stock solution of Sodium nitroprusside was added to give the following final concentration of Nitric oxide in the media - 0 M, 10 ^-6 M, 10 ^-5 M, 10 ^-4 M, 10 ^-3 M.
4. Four hours after induction, 100uL of the culture was added to each well of 96 well plate.
5. Varioskan LUX multimode reader was used to measure the GFP fluorescence: Excitation at 475 nm and emission at 545 nm.

Result

BBa_k381001 transformed E. coli shows high GFP expression in moderate nitric oxide concentration and decreased expression in both very low and very high NO concentration.

Mean GFP fluorescence (Excitation 494nm/Emission 525nm) of BBa_I13522 transformed E.coli induced by different concentrations of NO in the growth media are shown in the graph above.

Conclusion

The bacterial promoter PyeaR is inducible depending on the concentration of NO available in the media. It is induced best in the range of 10^-5 M to 10^-4 M.

Sequence and Features