Difference between revisions of "Part:BBa K216005:Experience"
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== NRP-UEA 2012 == | == NRP-UEA 2012 == | ||
| − | The team decided to develop the | + | 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). |
This characterisation included: | This characterisation included: | ||
| − | <br><br>-'''Growth studies''' of the | + | <br><br>-'''Growth studies''' of the PyeaR biobrick, the mammalian-bacterial (M-B) biobrick, and the bacterial-mammalian (B-M) biobrick. |
<br><br>-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. | <br><br>-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. | ||
<br><br>-Measuring the number of cells which fluoresce in different potassium nitrate concentrations using '''flow cytometry'''. | <br><br>-Measuring the number of cells which fluoresce in different potassium nitrate concentrations using '''flow cytometry'''. | ||
<br><br> | <br><br> | ||
| + | |||
==Growth Studies== | ==Growth Studies== | ||
Revision as of 15:42, 23 September 2012
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how you used this part and how it worked out.
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.
User Reviews
UNIQa875fbf590f79bdc-partinfo-00000000-QINU UNIQa875fbf590f79bdc-partinfo-00000001-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).
This characterisation included:
-Growth studies of the PyeaR biobrick, the mammalian-bacterial (M-B) biobrick, and the bacterial-mammalian (B-M) biobrick.
-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.
Growth Studies
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)
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 PyeaR, M-B and B-M. 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.
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.
