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-scale combined experiments: