Difference between revisions of "Team:USP UNIFESP-Brazil/Interlab"

Imagine you are a Biohacker or someone very interested in Science stuff, but you have no money… How could you avoid expensive high-end equipment and yet, still obtain some data about the promoters you love?

In order to do so and draw a sketch of our promoter’s strength we have followed and updated the 2015 USP_Brazil iGEM team approach for an inexpensive and quick analysis by taking digital photos and analyzing them on open-source softwares for image processing (GIMPand ImageJ).

All test devices and controls were grown on both solid (LB-Agar) and Liquid (LB and M9) media and photos were taken by a regular cellphone under the effect of fluorescent white or blue light lamps (for exciting GFP reporter molecules). The choice of comparing both LB and M9 liquid media was based on an extensive number of reports regarding the influence of auto fluorescence of LB on measurements. Thus, we wanted to have check if the outcome of this effect would be so strong that it could be visually detected.

On a direct analysis under the blue light lamp, we can observe that there is a huge difference between M9 and LB samples (Figure 2). While we can easily observe different degrees of GFP expression on M9, it is almost impossible to do so on LB due to its intrinsic fluorescence. Even though, on both media, TD1 seems to be the strongest promoter, followed by TD2, which is similar to PC and stronger than TD3 (easier to see on M9). The last test device, TD3, seems to behave very similarly to the negative control. To sum up, at a first glance, our promoter’s strength rank is:

In order to obtain more data about the relative promoter expression by macroscopic visual analysis, we have used the ImageJ 3D plugin (Interactive 3D Surface Plot) to create an intensity Heatmap based on photos of devices grown on liquid culture (M9) and on LB-Agar plates. As we can see in figure 4 and figure 5, the same ranking observed before persists. This method, together with the GIMP Image analysis, represent an easy, quick and inexpensive method for outlining promoter strength.

To sum up, we have shown that simple image analysis based on regular digital photos and open-source common softwares can provide an overview of the promoter strength on the macro scale. We have also shown the importance of choosing the correct media for your measurements, highlighting the high levels of noise found in LB media. We will now compare the fluorescence patterns found in this macroscopic and static analysis to the ones found in the study of dynamic GFP expression behavior over time, along the growth curve of a bacterial population.

Analysis of GFP expression on Fluorescence Spectrometer allowed us to observe the expression dynamics of our test devices on a population-scale. The data acquisition was performed in a Fluorescence spectrometer model LS55, Perkin Elmer. Samples were grown overnight on a shaker (37°C), diluted in cuvettes and measured for fluorescence every hour, during 6 hours and our cells were on exponential growth phase after 2 hours of growth. The spectrometer was configured for 250-500 nm excitation and 480-750 nm emission, using 10/10 slit and a filter 1% attenuation. The optical density (OD) was measured on a common spectrometer.

We have analysed GFP expression for FITC, TD 2, TD 3, PC and NC. At first, Test Device 1 was not present on our InterLab kit and was sent to us separately after a few weeks. As this experiment was done on a separate lab (as a collaboration between Brazilian Universities) before the arrival of TD1, the data for the following Device is not present. Furthermore, samples were incubated on ice for approximately 10 hours after absorbance quantification, prior to fluorescence measurement as the utilized fluorimeter was located on another city (yes, the guys travelled many miles just to get their samples measured!). This process may have generated abnormal behaviours, as the quantifications showed below are not directly comparable with the rest of our data. However, we can still get a grasp of the relative strength of the Test Devices with this measurement!

We have used the calibration solutions provided (LUDOX for Absorbance and FITC for fluorescence – figures 6 and 7) in order to help normalizing the data gathered from different laboratories. Furthermore, all fluorescence measurements compared emission wavelengths ranging from 480nm to 745nm in order to find the best output wavelength (figures 6 to 12). The data for Absorbance (OD600 - Figure 13), was used to calculate the GFP /OD600 rates for each time point for all test devices (figures 14 and 15)

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