Revision as of 03:31, 2 November 2017

Applications of BBa_K381001

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.

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iGEM11_UTP-Panama Experience: Cold Shock to BBa_K381001

Objectives:
1. Test the BioBrick operation to different substrates and temperatures, using KNO3 40 mM:
a.Substrates:
i.LB
ii.Minimal essential medium (MM)
iii.Saline solution (SS)
iv.Lunar Land (not used)
b.Temperatures:
i.8°C
ii.X°C (not used)
iii.23°C
iv.37°C
2. Improve the characterization of the BioBrick tested in other substrates to study the performance of the promoter.
3. Define the Biobrick promoter strength at the temperatures and selected substrates.

Methodology
We chose 4 temperatures: ´

• 5-10°C
  
• 20-30°C
  
• 37°C
  

These temperatures were selected so we could evaluate the response to cold shock in various ranges of our BioBrick. Since the lab equipments we had available couldn’t reach temperatures between 11-20°C this temperature was not used during the experience. Finally, the lab equipment allowed us to make the experiments in: 37°C, 23°C and 8°C.
We also chose 3 different Growth Media:

• Saline Solution
  
• Liquid LB
  
• Minimal Media
  

These growth medias where selected to evaluate the response of our BioBricks in different metabolic pathways. According to the characterization made by Team Edinburgh 2009, the Pyear promoter (BBa_K216005) used by Bristol 2010, works best in minimal media and complex media with a nitrate concentration of 40mM. Using this reference, we added 40mM of KNO3 to every growth media in the different temperatures for Bristol 2010 - BBa_K381001 and UTP-Panama 2011 – BBa_K672000.
We prepared 9 15mL Falcon Tubes, these tubes contained:

• First 3 (one for each temperature): 10mL of Saline Solution + a colony of the BioBrick + 40mM of KNO3.
• Second 3 (one for each temperature): 10mL of Minimal Media + a colony of the BioBrick BioBrick + 40mM of KNO3.
• Lat 3 (one for each temperature): 10mL of Liquid Luria Broth + a colony of the BioBrick BioBrick + 40mM of KNO3.
  

RESULTS

iGEM11_OUC Experience

Applications of BBa_K381001

From the beginning, we use the linearized plasmid backbone pSB1C3 in the 2011 spring DNA distribution. But the standardization with them all end up with failures.

Then, we began to seek for a new plasmid backbone. We found the K381001 in pSB1C3. If we cut the pSB1C3 with EcoR I and Pst I, we could get pSB1C3 plasmid backbone for standardization.

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The verification

The pSB1C3 is supposed to be 2049bp, however, it appears around the marker 3000bp. We have the pSB1C3 in K381001 sequenced and found its total length is 2944bp, and its total sequence is as below,

Through sequence analysis, we could find that the base in blue have a 99% homology with the right pSB1C3. It lacks a C and a A in the beginning. In the end, it has a 71 base homology with the right pSB1C3. The other sequence left do not accord with the right pSB1C3.

NRP-UEA 2012

The team (NRPUEA 2012) decided to develop the PyeaR biobrick (BBa_K216005) further by ligating it to its mammalian counterpart: CArG promoter sequence E9-ns2. The genes were synthesised in two orientations, bacterial-mammalian (BBa_K774000) and mammalian-bacterial (BBa_K774001) as initially we were not sure what effect gene order would have on gene activity. The aim of this development was to increase the flexibility of the PyeaR promoter so that it can be used in both mammalian and bacterial systems. This is something that we thought was important as sensing nitric oxide in the human body has a wide range of therapeutic applications (please see the future applications section on our wiki).

In order to begin to develop experiments to characterise the hybrid promoters + fluorescent proteins experiments were also carried out on a biobrick containing PyeaR + GFP (Part BBa_K381001, Bristol 2010). In these experiments transformed E. coli was inoculated into liquid culture, which in turn had varying potassium nitrate concentrations added to it. They were then left to grow before being spun down and viewed under a UV box in order to observe. The different concentrations of potassium nitrate that the transformed E. coli was grown in were: 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM.

A photograph of spun-down media containing potassium nitrate (to induce the promoter) and E. coli transformed by PyeaR + GFP (art BBa_K381001). Each sample was grown with a different concentration of potassium nitrate, from left to right: 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM.]]

The figure suggests that fluorescent proteins have been expressed by the bacteria grown in media containing potassium nitrate due to the fluorescence shown under the UV box. It also suggests that different concentrations of potassium nitrate correlate with different intensities of expression due to the observable differences in fluorescence as the tubes are viewed from left to right, going up the gradient. The negative control of 0 mM potassium nitrate appears to show no fluorescence, suggesting it is indeed the potassium nitrate that is inducing the promoter.

As part of the characterisation of our new biobricks, we thought that it would be interesting to compare the growth of our biobricks to the growth of the PyeaR+GFP composite.

The study involved testing the affects of transforming E.coli with different promoters on its growth over time. The promoters E.coli had been transformed with were the PyeaR+GFP promoter, the bacterial-mammalian promoter(BBa_K774000) and the mammalian-bacterial promoter (BBa_K774001). These are promoters which all react to nitrogenous species. By running these together, we can obtain a direct comparison between all three of these promoters on the growth of E.coli. To see if there are any significant changes, the study was run alongside E.coli cells which had not been transformed with anything. For the rest of this brief report, untransformed cells will be referred to as Alpha cells and the other E.coli cells with transformations will be referred to as the promoter with which they were transformed with.

The E.coli cells used in the study and for the transformation are the same type of cells (Alpha select gold standard cells from Bioline). A colony was inoculated into 5ml of LB media overnight and the cells spun down the following morning and diluted with fresh LB until an OD reading at 600nm of 0.2 ± 0.01 was obtained. Three repeats were made of each sample.

The study lasted for 12 hours. An OD reading at 600nm was taken once an hour. Between the hour, the cuvettes were put into a 37ᵒC incubator to encourage growth and for standardising measurements with other growth studies. To calculate the number of cells in the samples, a calibration curve was set up. This involved using cultures of the E.coli cells without transformations. The E.coli cells were diluted with different volumes of LB and OD readings were taken as well as plating on Agar plates. After a day of growth, the numbers on these plates were counted and recorded. The CFU/ml was calculated. When the OD readings (x axis) and the CFU/ml (y axis) readings are plotted, the equation of the line of best fit, gives a conversion for the absorbance readings. This allowed us to measure the growth. This is demonstrated in figure 1.

Figure 1. Calibration curve to calculate the conversion factor between OD reading at 600nm and the number of colony forming units growing per ml (CFU/ml)

We found that there was a significant difference between Alpha cells and PyeaR cells. Initially, Alpha cells had a greater growth rate, but after the third hour into the study, the growth rate of PyeaR was faster than that of Alpha cells. The overall growth rate of PyeaR cells was significantly faster that Alpha cells (Levenes Test, F = 1.009 p = 0.372; T Test, t = 4.196, df = 4, p = 0.014).

Figure 2. Growth of PyeaR transformed E.coli cells relative to Alpha cells (untransformed cells). Error bars show the standard deviation between the three repeats. For clarity reasons, lines of best fit are not shown The growth pattern and rate of E.coli cells with or without transformation with B-M and M-B show little difference. Any differences in growth rate were not significant. There was lots of overlap. As previously described, there was a significant difference between the growth rate of PyeaR and Alpha cells. There was also a significant difference between MB/BM and PyeaR cells. The statistical results can be seen in Table 1.





Figure 3.Growth over 12 hours of Alpha, M-B and B-M. Error bars and lines of best fit are not shown for clarity reasons.

Table 1. ANOVA readings of statistical differences between Alpha (1) PyeaR (2), MB (3) and BM (4).

From all the above graphs, it can be seen that with the starting concentration of cells as high as they are, the cultures are in exponential stage and do not undergo lag phase. A further growth study will be carried out on purely the lag phase with lower starting concentrations. As the starting absorbances here are approximately 0.2 at a wavelength of 600nm, the lag phase study will involve starting absorbances of 0.04 and lower.

= HFLS_H2Z_hangzhou 2017

BBa_K2346006 (https://parts.igem.org/Part: BBa_K2346006) is a functional improved device for this. We replaced the original reporter gene GFP with brighter reporter gene sfGFP, in the hope to improve the sensitivity for the device to detect nitrite and nitrate.

Conclusion We tested several environment for Pyear+sfGFP and compared it with Pyear+GFP. Our device compared with previous reporter BBa_K381001(https://parts.igem.org/Part:BBa_K381001) is significantly stronger in sensitivities. Furthermore, the sensitivity towards nitrite is greatly improved: just with a concentration of 1.5mM nitrite, sfGFP can give equal A.U. value compared with GFP device in 40mM nitrate. Furthermore, using sfGFP, we can draw distinction between 40mM and 20mM nitrate, previously not feasible with Pyear+GFP.

LZU-China 2014

Summary:
Our team means to use the part BBa_K216005 to detect the appearance of PNP(p-nitrophenol). So we use K381001 to perform the pre-experiment. We are so happy to find that this part,BBa_K381001, can express green fluorescence when PNP appears.

Methods:
We use the E.Coli DH5 alpha to be the host. The edited bacteria are cultured at 37 centigrade for 18 hours. We next mixed the bacterial liquid 10μl with a series of solutions and we went to see if there would be green fluorescence after 2 hours. We also developed a pathway which can make elctric current by producing riboflavin to sense the PNP. We detect the concentration of riboflavin after culturing in a series of liquid for 2 hours.

Results:

Figure-1 Fluorescence of different system. a.bacterial liquid with 10mM PNP;b.bacterial liquid with ddH2O;c.bacterial liquid with 10mM KNO3;d.bacterial liquid with 10mM KCl.

Figure-2 A sensor pathway we designed.

Figure-3 The riboflavin producing value on the increasing of PNP.

Discussion:

As you can see, the concentration of riboflavin is increasing with the increasing of PNP under 0.5mM. Then it decreased. A reason is that the PNP can kill the strain so they cannot produce riboflavin. While the strain always can live in the 20mM PNP so we should determine the real reason in the future.

iGEM15_HKUST-Rice Experience

HKUST-Rice team aims to create a NPK biosensor. To provide more characterization data on the nitrate-sensing promoter, we further characterize this promoter.

Result

After obtaining the quantitative results of GFP signal intensity using an EnVision® multilabel reader, the fluorescence signal were represented in fluorescence divided by biomass.

Dynamic range Characterization of P_yeaR in LB and M9

• GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 charaterization data obtained in 3 different days. Error bars were presented in SEM.

According to A, a plateau was shown starting from the 10 mM concentration point, suggesting that 10 mM nitrate concentration is the saturation point of P_yeaR and the dynamic range of P_yeaR is shown to be between 0-10 mM in our study. The relative fluorescence level increases 7.21 folds between 0 mM and 10 mM concentrations of nitrate.
According to B, a plateau was shown starting from the 500 μM concentration point, suggesting that 500 μM nitrate concentration is the saturation point of P_yeaR and the dynamic range of P_yeaR is shown to be between 0-500 μM in our study. The relative fluorescence level increases 3.12 folds from 0 μM and 500 μM nitrate concentrations.

UNebraska-Lincoln 2016

Summary:
Our team used the yeaR promoter in our project in order to create a nitrate-sensitive kill switch. In order to assay whether the promoter is sensitive at concentrations below 150 uM (the "dangerous" nitrate concentration threshold in natural waterways), we characterized K381001 at a variety of concentrations, lower than had previously been tested. We created a fluorescence (rfu) vs Nitrate concentration graph, in order to determine it's effective activity at our select concentrations. We also determined the activity of K381001 cells over time.

Methods:
We transformed K381001 into competent Genehogs cells, plated them, and incubated the plate at 37ºC for 18 hours. We inoculated these transformed cells into a 5 mL sample of LB Broth with 5uL cm34, and placed the culture in a 37ºC shaker for 18 hours. The overnight culture was then subcultured into a 96 well plate (1 mL LB broth, 1 uL cm34, and 10 uL of culture), the 96 well plate was placed in the 37ºC shaker until the OD600 of the subcultures reached 0.4-0.6. Each subculture was then induced with a different concentration of potassium nitrate (triplicate measurements). The 96 well plate was then placed in the 37ºC shaker for 18 hours. Each sample was pelleted and resuspended in phosphate buffer and the absolute fluorescence and OD600 of each sample was measured. This allowed a quantitative determination as to the effectiveness of the yeaR promoter at varying nitrate levels.
We conducted further testing, we followed the same procedure to prepare our samples, but we only tested 5 different concentration. The absolute fluorescence and the OD600 were measured every half hour for four hours in order to test the GFP expression over time.
For more detail on our exact procedures, you can view our full lab manual at: http://2016.igem.org/Team:UNebraska-Lincoln/lab_manual" Our full procedure can be found on page 20.

Results:

Figure 1: Our full characterization of K381001.

Here, you can see the full graph of all our triplicate measurements. In order to interpret our data, we selected four ranges, and displayed the average absorbance values in order to see if the activity of the BioBrick noticeably increased with concentration of nitrate ion. We conclude that the change in activity of K381001 is not discernible at concentrations below 10 microMolar nitrate ion. This contributed to the characterization of this part, and helped us determine that it would not be feasible to create a kill switch that was sensitive at concentrations below 10 uM. Fortunately, the threshold of dangerous nitrate levels is much higher at 150 uM.

Figure 2: Our characterization of K381001 from 0 uM to 2uM, at increments of 0.25 uM.

Figure 3: Our characterization of K381001 from 2uM to 10uM, at increments of 2uM.

Figure 4: Our characterization of K381001 from 10uM to 100 uM at increments of 20uM.

Figure 5: Our characterization of K381001 from 100uM to 1000uM at increments of 200uM.

Figure 6: Our full time-based characterization of K381001.