Part:BBa_K774000:Experience

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NRP-UEA-Norwich 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 (Scott, S.D. et al.2002). To provide additional restriction enzyme sites that may become useful during later cloning steps, BamHI, HindIII and NdeI were added between the 2 promoters. 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 could 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 (please click here.)

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 graph below shows the mean average of all recorded 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, B-M/M-B and PyeaR transformed cells are shown in order from highest to lowest trendlines. One single trendline was used to represent B-M and M-B because the trendlines were extremely similar. Using the initial concentrations of 0.3±0.1 it can be seen 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.

Ligating the bacterial-mammalian promoter to fluorescent proteins

The hybrid promoter was ligated to two different reporters: enhanced Cyan Fluorescence Protein (eCFP)(BBa_K774004) and Red Fluorescent Protein (RFP) (BBa_K774005). The hybrid promoter was characterised by observing expression of flourescent protein, and found to have increased transcription in response to increasing concentrations of potassium nitrate.

NOTE: All data for the fluorometer has had the equivalent 0 mM reading subtracted from it in order to nulify the affects of light scattering due to cell debris

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 0mM 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 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. 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.

Flow Cytometry

Flow cytometry was used with part BBa_K774005 to quantify the number of cells which fluoresced in response to induction by potassium nitrate.

Three tubes of media were inoculated with E. coli transformed by the B-M + RFP biobrick (BBa_K774005). Each tube then had potassium nitrate added to it at different concentrations; 0 mM, 1 mM and 10 mM respectively. The E. coli were grown over night and then spun down, fixed in 4% PFA and re-suspened in 500ul PBS. The samples were then analysed in an Acuri C6 or BD Aria II flow cytometer.

[http://2012.igem.org/Team:NRP-UEA-Norwich/Protocol Full Protocol]

Flow cytometry data for B-M RFP transformed E. coli that were grown in either 0 mM, 1 mM or 10 mM potassium nitrate. Top row: Scatter plot of raw data and gating strategy utilised. Middle row: RFP Fluorescence profiles of samples. Lower left: Fluroescence profiles of the three samples overlai on the same plot.

As the graph indicates, the number of cells which fluoresce is proportional to the concentration of potassium nitrate that the cells are exposed to. Suggesting that our hybrid promoter is functional as expected.

Refernces

Scott, S.D., Joiner, M.C. & Marples, B., 2002. Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy. Gene therapy, 9(20), p.1396-402. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12365005 [Accessed June 24, 2012].

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