Part:BBa_K216005:Experience
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Applications of BBa_K216005
Edinburgh iGEM team, October 2009: To test this part, it was combined with lacZ minigene J33202 for Miller assays (giving part BBa_K216009), and also with GFP for analysis using the promoter characterization kit of Jason Kelly. Results from overnight induction with Miller assay indicated stronger induction by nitrate than nitrite. A time course of induction using the GFP construct also indicated that nitrate gave stronger induction in the first few hours.
Results from Miller's Assay
Results from GFP promoter characterization kit
PyeaR response to nitrates
Jason Kelly’s assay demonstrates that our promoter is sensitive to nitrates. The most sensitive time period is between 5 ½ hours (320 minutes) 7 ½ hours (440 minutes) after induction. The sensitivity decreases with time, but induction is still at a high level after the peak activity of the promoter. Only two concentrations are shown (0 mM and 10 mM), as we have demonstrated that 10 mM is the sensitivity peak of this promoter (look at last graph).
PyeaR response to nitrites
Jason Kelly’s assay demonstrates that our promoter is sensitive to nitrites. The fluorescence levels begin to build up only after about 8 hours; therefore other irrelevant results are omitted from this graph. The sensitivity curve in this case is less steep compared to the one produced due to the presence of nitrates. This indicates that the promoter responds differently to these two nitrogenous compounds. Again, only two concentrations are shown (0 mM and 10 mM), as sensitivity peaks at 10mM.
PyeaR response to nitrates and nitrites after overnight induction
Here we can compare the activity of this promoter to different concentrations of both nitrates and nitrates. We observed that the promoter is activated more readily by nitrates compared to nitrites. Nevertheless, the activation produced by nitrites remains significant. The peak activation concentration for both compounds is 10 mM, after which activation efficiency decreases.
UCL 2015: BBa_K381005 Testing
The PyeaR promoter (BBa_K216005) from the 2009 Edinburgh iGEM team was the part we wanted for our Nitric Oxide sensing device. To test if this promoter works, we used the BBa_K381001 (BBa_K216005 + BBa_E0840) part made by 2011 BCCS-Bristol iGEM In order to test the promoter, we transformed the DH5-α with BBa_K381001 and measured the fluorescence with a plate reader after being induced with varying concentrations of potassium nitrate (0 mM - 40 mM) at OD = 0.4.
Figure 1. Fluorescence of cultures when PyeaR promoter was induced at varying concentrations of potassium nitrate.
Fig. 1 shows that at after around OD = 0.4, fluorescence becomes higher in the cultures containing potassium nitrate compared to the culture with no nitrate. On another note, no large increase in fluorescence was seen from increasing the concentration of potassium nitrate beyond 5 mM, suggesting that 5 mM potassium nitrate is enought to induce the promoter to maximum strength. Surprisingly, the cultures with 5 mM and 10 mM potassium nitrate had a generally stronger induction compared to the 25 mM and 40 mM cultures.
User Reviews
UNIQ6b9c4926ae440218-partinfo-00000001-QINU UNIQ6b9c4926ae440218-partinfo-00000002-QINU
NRP-UEA 2012
The team decided to develop the PyeaR biobrick 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).
The characterisation of our biobricks was carried out as follows:
-Growth studies of the PyeaR biobrick (BBa_K381001), the mammalian-bacterial (M-B) biobrick (BBa_K774001), and the bacterial-mammalian (B-M) biobrick (BBa_K774000).
-Measuring the fluorescence of the M-B biobrick ligated with Red Flourescent Protein (RFP) and enhanced Cyan Fluorescent Protein (eCFP), as well as the B-M biobrick ligated with RFP and CFP, in response to induction with different potassium nitrate concentrations.
-Measuring the number of cells which fluoresce in different potassium nitrate concentrations using flow cytometry.
Transfecting part BBa_K774006 (mammalian-bacterial promoter ligated with eCFP) into mammalian cells to detect fluorescence and determine the functionality of the promoter.
Growth Studies
A comparison between the growth of E.coli cells, before and after transformation with the bacterial-mammalian promoter, as well as the mammalian-bacterial promoter (BBa_K774001) and PyeaR + GFP composite (BBa_K381001))
The study involved testing the affects of transforming E.coli with different promoters on its growth over time. The promoters E.coli was transformed with were PyeaR, M-B and B-M. These promoters all react to nitrogenous species. By running these growth studies together, we were able to obtain a direct comparison between all three of these promoters on the growth of E.coli. To see if the presence of novel promoters caused any significant changes in growth, 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 which have been transformed will be referred to as the promoter with which they were transformed with.
The E.coli cells used in all studies are Alpha select gold standard cells from Bioline, which have a hight transformation frequency.
To begin, a colony was inoculated into 5ml of LB media overnight, 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 to standardise measurements across all of the growth studies. To calculate the number of cells in each sample, a calibration curve was set up. This involved using cultures of the E.coli cells which had not been transformed. The E.coli cells were diluted with different volumes of LB and OD readings were taken at the same time 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 were 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 M-B/B-M 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.
A comparison between the growth of E.coli cells, before and after transformation with PyeaR + GFP (BBa_K381001) and B-M and M-B (in pSB1C3)- Lag Phase Study
Following the above study, we found that a lag phase only study needed to be carried out to see if there was a significant difference in the lag phase. Again the study protocol was the same except that the starting concentration absorbances at 600nm was lowered to <0.04. It was extremely difficult to keep the absorbances ranges within 0.005 so the range is actually 0.3±0.1. The below graph shows the mean average of all the data; using the data from the calibration curve, the absorbances were converted to colony forming units per ml (CFU/ml). The trend lines of alpha cells, BM/MB and PyeaR transformed cells are shown within this order from highest to lowest trendlines. One single trendline was used to represent BM and MB because the actual trendlines were extremely similar. Using the initial concentrations of 0.3±0.1 showed that there is little difference between the growth rates. Using statistical analysis, it was found that there was no significant difference between any of the transformed cells relative to Alpha cells or to each other (Anova, p > 0.05).
From this study we have found that changes in growth occur during exponential growth phase and not the lag growth phase.
Using reporters: Red Fluorescent Protein (RFP) and enhanced Cyan Fluorescent Protein (eCFP)
The two hybrid promoters were ligated with RFP (BBa_K774007 and BBa_K774005)and CFP (BBa_K774004 and BBa_K774006) in order to characterise them further.
This image (right) shows competent cells transformed with part: BBa K774004 and grown in media containing potassium nitrate (as a source of nitrates in order to induce promoter activity) at concentrations of 0 mM, 10 mM, 50 mM and 100 mM (from right to left). The E. coli was grown for 6 hours and then added to eppendorf tubes and spun down in a centrifuge in order to produce a pellet. The four samples were then viewed under a UV box to assess for fluorescence; as the photograph to the right shows that the sample at 0 mM potassium nitrate did not fluoresce, however those at 10, 50 and 100 mM potassium nitrate did fluoresce. They also appeared to fluoresce at the same strength, suggesting that 10 mM was equal to or above the maximum sensitivity level of this part.
The graph above shows the flourescence measured from the expression of eCFP due to the response of the bacterial-mammalian promoter to different concentrations of potassium nitrate. The wavelength reading which corresponds to eCFP is between 440-500nm. The graph clearly demonstrates that between 0mN and 15mM there is a proportional relationship between fluorescence intensity and potassium nitrate concentration. There appears to be a sharp increase in fluorescence intensity between 5mM and 10mM, and the rate at which intensity increase gradually decreases so that there is only a small increase between 15mM and 20mM.
The graph above shows the flourescence measured from the expression of eCFP due to the response of the mammalian-bacterial promoter to different concentrations of potassium nitrate. The wavelength reading which corresponds to eCFP is between 440-500nm. The graph clearly demonstrates that between 0mN and 15mM there is a proportional relationship between fluorescence intensity and potassium nitrate concentration. It can be noted that at a 20mM concentration the intensity of fluorescence sharply decreases back down to the level of 5mM potassium nitate concentration. This may be due to the cell overexpressing eCFP up to the point at which the excess protein begins to form inclusion bodies which can no longer fluoresce; alternatively, this could be due the potassium nitrate concentration reaching the critical concentration at which it becomes toxic to the cell. This data differs to the readings taken from the bacterial-mammalian promoter ligated to eCFP, as well as the hybrid promoters to RFP, which may suggest there is a difference in the molecular mechanisms that these promoters function by; however at this point the change in intensity at 20mM is inconclusive and is an area which we would like to look into further.
We were initially unsure of the effect that the orientation of the bacterial (pYEAR) and the mammalian (CaRG) genes would have in gene expression, therefore we synthesised two hybrid promoters in the orientation bacterial-mammalian and mammalian-bacterial. The graph above compares the intensity of fluorescence of the two hybrid promoters (BBa_K774004 and BBa_K774006) ligated to eCFP. There is a distinct difference between the intensity of fluorescence produced by the bacterial-mammalian promoter and the mammalian-promoter which is something that we would like to look into further. It is particularly interesting that at an intensity of 109a.u. the mammalian-bacterial promoter returns to the same level of intensity as the apparent maxiumum of the bacterial-mammalian promoter at 40a.u.
We also ligated both of our hybrid promoters to Red Fluorescent Protein (RFP), and the results can be seen below.
The graph above shows the flourescence measured from the expression of RFP due to the response of the bacterial-mammalian promoter to different concentrations of potassium nitrate. The wavelength reading which corresponds to RFP is between 600-650nm. The graph clearly demonstrates that between 0mN and 15mM there is a proportional relationship between fluorescence intensity and potassium nitrate concentration. A similar pattern can be seen here as for the mammalian- bacterial promoter with eCFP as at a 20mM concentration the intensity of fluorescence sharply decreases, however the intensity here decreases down to a level between 10mM and 15mM potassium nitate concentration. There is also only a small difference between 5mM and 10mM potassium nitrate, which differs to the pattern seen with the bacterial-mammalian promoter ligated to eCFP. As previously stated, this may be due to the cell overexpressing eCFP up to the point at which the excess protein begins to form inclusion bodies which can no longer fluoresce; alternatively, this could be due the potassium nitrate concentration reaching the critical concentration at which it becomes toxic to the cell. This data differs to the readings taken from the bacterial-mammalian ligated to eCFP, as well as the hybrid promoters to RFP, which may suggest there is a difference in the molecular mechanisms that these promoters function by; however at this point the change in intensity at 20mM is inconclusive and is an area which we would like to look into further.
The graph above shows the flourescence measured from the expression of RFP due to the response of the mammalian-bacterial promoter to different concentrations of potassium nitrate. The wavelength reading which corresponds to RFP is between 600-650nm. The graph clearly demonstrates that between 0mN and 15mM there is a proportional relationship between fluorescence intensity and potassium nitrate concentration. It has been found that for all biobricks apart from the mammalian-bacterial promoter ligated to eCFP at a 20mM concentration the intensity of fluorescence sharply decreases.
As previously stated, we were initially unsure of the effect that the orientation of the bacterial (pYEAR) and the mammalian (CaRG) genes would have in gene expression, therefore we synthesised two hybrid promoters in the orientation bacterial-mammalian and mammalian-bacterial. The graph above compares the intensity of fluorescence of the two hybrid promoters (BBa_K774007 and BBa_K774005) ligated to RFP. There appears to be no pattern if the difference between the intensities of these two promoters; however both promoters do show a decrease in intensity at 20mM potassium nitrate and decrease from a maximum intensity of 82a.u. (bacterial-mammalian) and 66a.u. to approximately 36a.u.
Flow Cytometry
Flow cytometry was used to quantify the number of cells which fluoresced in response to induction by potassium nitrate for both the B-M promoter ligated to RFP (BBa_K774005) and the M-B promoter ligated to eCFP (BBa_K774006). Both graphs indicate that the number of cells which fluoresce is proportional to the concentration of potassium nitrate that the cells are exposed to.
Left: Bacterial Mammalian promoter ligated to RFP
Right: Mammalian-Bacterial promoter ligated to eCFP
Flexibility of the hybrid promoter: Mammalian Cells
The hybrid promoterswere created to increase the flexibility of chassis a promoter can be used in. To fully characterise the functionality of B-M and M-B, it was therefore important that the promoter could in fact work within mammalian and bacterial chassis. As the above studies show, M-B and B-M both function in bacterial cells.
To incorporate B-M and M-B into a mammalian system, transformations were not possible, instead a transfection was carried out. Unlike the growth studies, where growing colonies were sufficient to prove that the DNA within plasmids were incorporated, reporter proteins: CFP and RFP were attached to allow visual characterisation.
To transfect the cells we used lipid based transfection. To do this, media, transfection reagent, nitric oxide donor, DNA and cells were required. Below is the full list of reagents used:
. DMEM (Dulbecco’s Modified Eagle Medium) without serum to transfect and later treated with DMEM with serum and 10% Fetal Calf Serum. Cells are transfected without serum as serum interferes with the process but cells can die without serum so later is treated with serum.
. M-B +CFP was the DNA used. More than one sample was used. From nanodropping the concentrations of DNA were found to be both 500nm/µL. To transfect, 6.5 µL of DNA was used. The exact DNA we used was labelled MB2-C11a and MB2-C12a. The DNA was obtained from different colonies from the same plate.
. SNAP (S-Nitroso-N-Acetylpenicillamine) at a final concentration of 500µM. SNAP is the nitric oxide donor. Unlike bacterial cells where potassium nitrate could be used as a direct source. SNAP is metabolised by cells to produce NO which then induces the BioSensor.
. LipoD293 which is the transfection agent. This creates a membrane around the DNA which then binds to the mammalian cell and allows entrance of DNA into cell. This is much like endocytosis.
. The chassis used was MCF7 which is a human breast cancer cell line. The cells (30 µL) were seeded into a 6 channel slide at a concentration of 3 x 105 cells/ml.
In the process of transfection, LipoD293 was mixed with DNA at left for 15mins. Meanwhile, the media was removed from the cells and washed with serum free DMEM (100 µL). The transfection mixture was then added to the relevant channels on the slide. Below is how our channels were labelled.
For the full transfection protocol please click [http://2012.igem.org/Team:NRP-UEA-Norwich/Experiments here]
After the transfection, the media was changed to media containing serum and also SNAP was added. The cells were then viewed and the following images were obtained.
The images show what looks like exclusion bodies which have a greater concentration of fluorescent proteins than within cells. In the control without SNAP, there are none of these exclusion bodies found. In the control photo with SNAP and also the transfected cells without SNAP added, there are a few exclusion bodies. However, in the photos that showed transfected cells with SNAP added, there are a large number of these.
We do not know for certain what these may be, but a possibility is that the transfection was successful and the MB promoter does work. It may be that due to MB attachment to fluorescent proteins, the cells are producing exclusion bodies to rid the cells of these. In the control with SNAP, the cancer cells react to NO in the human body. These may be exclusion bodies formed from cells in general in reaction of NO. As to the transfected cell with exclusion bodies, there are very few of these. MB may be very sensitive to NO. As NO is naturally produced by cancer cells to induce angiogenesis, these may be for that reason.
Another possibility is that NO has induced the cells to apoptose and this has lead to vesicles forming containing the fluorescent proteins (Yu, et al., 1999). In non transfected cells, there is less fluorescent proteins compared to the transfected cells and hence there are more fluorescing vesicles.
Following transfection, to test the cytotoxicity of NO, the number of cells after addition of SNAP was calculated. The MCF7 cells were seeded into 6 well plates again at a concentration of 2.5 x 105 cells/ml. SNAP was then added at 500µM 2 days after plating. The cells were counted 24 hours after the addition of SNAP. For the full cell count protocol please click [http://2012.igem.org/Team:NRP-UEA-Norwich/Protocol#Cell_Counting here].
From previous studies such as that by Lala and Chakraborty, 2001 have shown that NO can lead to cytostasis and apoptosis. Our assay further confirmed this.
References
Lala, P.K. and Chakraborty, C. (2001) 'Role of nitricoxide in carcinogenesis and tumour progression', The Lancelet, 2;149–156.
Yu, W., Simmons-Menchaca, M., Gapor, A., Sanders, B.G. and Kline, K. (1999) 'Induction of Apoptosis in Human Breast
Cancer Cells by Tocopherols and Tocotrienols', Nutrition and Cancer, 33;26-32.