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Characterisation Experiments

Growth Studies

A study of growth of PyeaR in different concentrations of potassium nitrate

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 cell (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.

Functionality Studies

Flexibility of the hybrid promoters: BM and MB

The hybrid promoters, BM and MB were created to increase the flexibility of chassis a gene system can be inserted into; granted this is not a complete system but only a promoter. It was therefore imperative that the for this to be a fully functional promoter in the sense it that it does what it was created to do, it must work in both mammalian and bacterial chassis.

Characterisation of BM and MB in different concentrations of potassium nitrate

Following the experiment measuring the affect on the intensity of GFP fluorescence of PyeaR-GFP at different concentrations of potassium nitrate, a study on how different levels of potassium nitrate affect fluorescence intensity of BM and MB attached to fluorescent proteins was tested. Where previously the study was carried out in a qualitative fashion, this was carried out to collect data quantitatively using a fluorometer. Overnight cultures of BM and MB attached to RFPs and CFPs containing different levels of potassium nitrate prepared. Using 5ml of the cultures, cells were peletted. The overnight cultures contained different concentrations of potassium nitrate (0mM, 5mM, 10mM, 15mM and 20mM). These cells were then resuspended and lysed (full protocol on protocol page). These cultures were then diluted using Tris buffer (0.5ml sample to 1.5ml buffer). Readings from the fluorometer wavescans were recorded.

Comparing BM and MB with CFP fluorescence showed that BM-CFP intensity is relatively low when compared to MB. MB seems more sensitive to nitrate levels than BM. However, for both, it seems that once a threshold of nitrate levels is reached, there is not additional expression. Beyond this concentration, the intensity drops. This may be due to a number of factors. The cells may die due to overload of proteins which may have a toxic affect. For BM this threshold seems to be just above 20mM and for MB this appears to be around 15mM. This is seen in the figure below.

Comparing BM and MB with RFP fluorescence showed a similar result to the CFP study. However, both BM and MB have similar sensitivities to potassium nitrate levels, BM seems to be more sensitive to potassium nitrate concentration this time. From this study it is clear that beyond a certain concentration of nitrates, the fluorescence intensity stops increasing and decreases instead. Again, this may be due to the capacity or tolerance of the cell to hold the volume of fluorescence. protein.

Transfection of BM and MB into mammalian cells

Test of the effect transforming E.coli with construct 1 and construct 2 has on the rate of growth

The team plan to carry out a growth study similar to that above for the BM MB, as when working with construct transformed e.coli team members have noticed quick growth . This will allow analysis of any differences in the rate of growth between E.coli transformed with either constructs from the alpha gold control.