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

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-level scale combined experiments

In order to best characterize the Biobricks, we have done experiments with differential sensibility thresholds, ranging from macroscopic analysis of photos taken by a cellphone camera to single cell analysis in a flow cytometer. The main rationale was to compare methods focused on different scales of the same system, providing both general and specific information about the behavior of the selected promoters. Furthermore, we wanted to try new inexpensive methods as regular macroscopic image analysis (taken by a cellphone, for example) and DIY-fluorimeter analysis. We present below an overview of those multi-scale approaches chosen by our team during the InterLab, from the macro to the cellular microscopic view:

• Macroscopic Analysis: Cellphone Photos quantitatively analyzed by GIMP and ImageJ open-source software
• Population Analysis I: DIY Fluorimeter based Assay
• Population Analysis II: Plate Reader Assay and Relative Promoter Units (RPU) calculation
• 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 (https://2015.igem.org/Team:Brasil-USP/interlabstudy) for an inexpensive and quick analysis by taking digital photos and analyzing them on open-source softwares for image processing (GIMP - https://www.gimp.org/ - and ImageJ - https://imagej.nih.gov/ij/).

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:

$$TD1 > TD2 = PC > TD3 = NC$$

In order to obtain more reliable and quantifiable data we have processed and analyzed our photos using the popular image editor GIMP (GNU Image Manipulation Program), following the 2015 USP_Brazil iGEM team protocol (https://2015.igem.org/Team:Brasil-USP/interlabstudy). We have generated intensity histograms for each device compared on Figure 3. As observed, the intensities ranking are comparable to what we have seen on our first analysis.

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.