Team:USP UNIFESP-Brazil/Interlab
AlgAranha Team USP-UNIFESP BRASIL
In the twelfth iGEM edition happens the third Interlab Study. This study is based on the characterization of standard biological parts and, as standard parts, it is fundamental to observe reproducibility and repeatability on their behaviour. For instance, even well characterized promoters in a given strain of E. coli may behave reasonably different in another strain. Acknowledging this challenge, the Interlab Studies is a way to gather experiments from all around the world and provide a more unified understanding about the fundamental building blocks of Synthetic Biology. Until last year, each research team had its own strains, plasmids and protocols, however, in an attempt to standardize the obtained data, specific protocols and calibration samples were provided for each iGEM team attending the Interlab 2016. With this approach, we can construct a rich knowledge base of standard biological parts, together with several study cases of different protocols and other details. The value this have to the whole community of Synthetic Biology is beyond doubt.
We have done not only the standard plate reader, cuvette-based and flow cytometry assays, but also tested for better measuring conditions (LB and M9 media) and for alternative methods ranging from DIY ones (digital camera and fluorimetric-based methods) to single cell analysis by fluorescence microscopy. We have also evaluated the promoter strength of all devices by Relative Promoter Units [2] using DH5α E. coli harbouring all devices and controls. Results show interesting differences: Device 2 (J23106) shows half the strength it would be expected in the original library. Thus, we have fulfilled both the InterLab study the extra credit requirements by searching for optimized measurement protocols and generating new cheaper and accessible approaches for assessing promoter strength.
Test Devices and controls:
We have received three Test Devices and one positive control derived from the Anderson’s library, a constitutive promoter library generated by single mutations, which affected the promoters’ strength in different ways. The devices are a combination of the Anderson’s promoters, RBS, a GFP reporter gene and a terminator. The negative control consist only on an inert sequence derived from the TetR operator. All devices and controls have the pSB1C3 plasmid (high-copy number) as backbone. Following the iGEM protocol, all plasmids were transformed into DH5α E. coli cells - following the iGEM transformation protocol - which were used as samples for all the different experiments. You can find more information about the devices below and on figure 1.
Positive control (PC) - I20270 in pSB1C3
Negative control (NC) -R0040 in pSB1C3
Test Device 1 (TD1) - J23101.B0034.E0040.B0015 in pSB1C3
Test Device 2 (TD2) - J23106.B0034.E0040.B0015 in pSB1C3
Test Device 3 (TD3) - J23117.B0034.E0040.B0015 in pSB1C3
Multi-scale combined experiments:
- Macroscopic Analysis: Cellphone Photos quantitatively analyzed by GIMP and ImageJ open-source software
- Population Analysis I: Cuvette-based measurements on Fluorescence Spectometer
- Population Analysis II: Plate Reader Assay and Relative Promoter Units (RPU) calculation
- Population Analysis III: Do-It-Yourself Fluorimeter-based Assay (Not finished yet. However, you can see our progress with the hardware development HERE (link))
- Sub-Population/Single Cell Analysis: Flow Cytometry Assay and Relative Promoter Units (RPU) calculation
- Microscopic Analysis/Single Cell: Fluorescence Microscopy and quantitative analysis by ImageJ
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:
Figure 2 Comparison between fluorescence of Test Devices on both M9 and LB. While M9 allows us to easily compare fluorescence intensities the same is not true for LB samples due to its auto fluorescence effect.
Figure 3 Green Intensity for each Test Device measured by GIMP. All measures from LB present similar values due to auto fluorescence effect.
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.
Figure 4 A heatmap of fluorescence intensity generated by ImageJ. Above: M9 liquid cultures with all devices were grown for 16 hours, photographed under a blue light lamp and transformed into a heatmap on ImageJ; Below: The heatmap can be graphically represented by a 3D interactive surface plot.
Figure 5 A heatmap of fluorescence intensity generated by ImageJ. Above: Single colonies for each device were streaked on an LB-Agar plate, grown for 16 hours, photographed under a blue light lamp and transformed into a heatmap by ImageJ; Below: The heatmap can be graphically represented by a 3D interactive surface plot.
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)
Figure 6. Fluorescence emission of FITC between 480-700nm wavelengths. The concentrations for FITC are the same as standard curve. The FITC1-m has the higher concentration while FITC12-m the lower concentration.
Figure 7. FITC standard curve
Figure 8. Fluorescence emission of NC between 480-700nm wave-length.
Figure 9. Fluorescence emission of PC between 480-700nm wave-lenght.
Figure 10. Fluorescence emission of TD2 between 480-700nm wave-lenght.
Figure 11. Fluorescence emission of TD3 between 480-700nm wave-lenght.
Figure 12. Fluorescence of all test devices (on MEFLs – normalized by FITC)
Figure 13. Abs600 measured for each Test Device
Figure 14 Fl/Abs600 for each device on LB medium with respective error bars. We can notice aberrant error bars for devices with low/absent promoter strength .
Figure 15. GFP/OD600 values for each device. We can notice the negative values for NC and TD3.
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