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Applications of BBa_K774000

A hybrid of the PyeaR promoter (BBaK216005) and the E9-ns2 CArG promoter (Ref:1). Two versions will be synthesised, with the promoters altering their position in relation to the 5’-end of the sequence.

To provide additional restriction enzyme sites that may become useful during later cloning steps, BamHI, HindIII and NdeI have been added between the 2 promoters.

It is envisaged that any open reading frame (e.g. RFP or GFP) will be cloned “downstream” (i.e. at the 3’-end) of these promoter sequences.

Ref:1

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].

NRP-UEA 2012

The aim of ligating the PyeaR biobrick with its mammalian counterpart: CArG promoter sequence E9-ns2 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.

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). 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 readings, the cuvettes were placed into a 37ᵒC incubator to encourage growth and also to standardise measurements between 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 which had not been transformed for a negative control. The E.coli cells were diluted with different volumes of LB and OD readings were taken at the same time as plating the samples 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.

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