Part:BBa_K381001:Experience
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.
User Reviews
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EliasGrimaldo |
The part work well under cold shock stress (20°C to 30°C) of 10-12 hr. |
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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.
User Reviews
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DonQuixia |
When we use the pSB1C3 for standardization, we found that the pSB1C3 have the right antibiotic resistance but a wrong DNA length. |
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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.
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.