Team:UNICAMP-EMSE Brazil/Results

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Revision as of 17:41, 28 September 2011

Overview

Functional tests

The students assembled the devices subparts: the SoxR/SoxS sensor (Device 2, NO sensor device/ IL-10 producer), the Adrenaline sensor (Device 1, CA/AI-3 sensor device/ IL-12 producer), and the hemolysin secretion system (Device 3, Protein Secretion System). All the sensors were also built with GFP downstream of the sensor itself, replacing the Device products: IL-12 at Device 1 and IL-10 at Device 2.

Device 2 testing: SoxR/SoxS system regulating GFP production

The Device 2 is a modification of Stanford team anti-inflammatory device (iGEM 2009 - https://2009.igem.org/Team:Stanford/ProjectPage), which comprises SoxR gene (BBa_K223047) under control of a Constitutive Promoter (BBa_J23119) and SoxS promoter (BBa_K223001 – deleted part), coupled to human IL-10 gene (sequence designed by the team, improved for bacterial expression).

In order to test the ability of Jedi Bacteria in sensing NO levels and activating genes linked to SoxS promoter, we built a Device testing, with GFP linked to SoxS promoter, as it is shown in the following schema (Figure 1):

Figure 1: Testing Device 2 through replacement of IL-10 to GFP.

The ability to recognize NO (nitric oxide), an inflammation signal molecule, was characterized for SoxR/SoxS sensor, and found to be FUNCTIONAL. In the section below we present the detailed results.

Figure 2: SoxS/SoxR fluorescence data for concentrations 0, 5, 10, 20, 30 and 40 µM of inducer.

The NO is a product related to inflammatory response in vivo. To test the sensor in vitro, we used http://en.wikipedia.org/wiki/Paraquat Paraquat as inducer, a molecule which imposes superoxide stress within the cell in a similar manner as nitric oxide.

The experiment indicated that our sensor was capable of inducible production of GFP (or another protein controlled by the sensor). In addition, the protein induction can be modulated through varying the inducer concentration (as shown by Figure 2). Higher concentrations of Paraquat exhibited higher fluorescence levels, which indicates increased GFP concentrations.

The experimental concentrations of Paraquat did not show significant differences in cell growth as shown by OD levels in Figure 3. The Stanford team (iGEM 2009) showed that Paraquat can be toxic to E. coli cells, with growth inhibition at 60 – 80 µM.

Figure 3: SoxS/SoxR optical density data for concentrations 0, 5, 10, 20, 30 and 40 µM of inducer.

@@ Line 74: / Line 74: @@
 Q[19] = "<i> Yeah, those dresses deserve us!</i> Marc Emery (UNICAMP-EMSE team student) about Marianna's dresses"
 Q[20] = "<i> I am really starting getting worried with you guys!!</i> Iolanda Albuquerque (UNICAMP-EMSE team advisor)"
+Q[21] = "<i> Relationship is not like those colorful things where everything fits perfectly ... The name of that is LEGO!</i> Izabella's Mom (mother of one of UNICAMP-EMSE team student)"
+Q[22] = "<i> Nice to meet you, I am Marianna</i> Marianna Favaro (UNICAMP-EMSE team student, said after Thibault cut his hair and become unrecognizable)"
+Q[23] = "<i> Who is this guy?</i> Danieli Gonçalves (UNICAMP-EMSE team student, said after Thibault cut his hair and become unrecognizable)"
+Q[24] = "<i> That guy is your friend?</i> Izabella Neshich (UNICAMP-EMSE team student, about Thibault, after he cut his hair and become unrecognizable)"
+Q[25] = "<i> But Nemaila said me that it was good!</i> Thibault Sabattier (UNICAMP-EMSE team student)"
+Q[26] = "<i> That guy is Caua Reymond</i> Marco Marinho (UNICAMP-EMSE team advisor said after Thibault cut his hair and become unrecognizable)"
+Q[27] = "<i> Marc don't put your head inside the flux or try to tan yourself in the UV transilluminator</i> Nemailla Bonturi (UNICAMP-EMSE team advisor warning the little student Marc Emery)"
+Q[28] = "<i> Piri-piri-piri-pi-pi-piri-pi (UNICAMP-EMSE team students Izabella, Danieli, Marianna, Juliana and advisor Nemailla singing a traditional song)"
+Q[29] = "<i> Everythings works at the end. If it has not worked, it's because we have not arrived to the end (UNICAMP-EMSE team students Izabella, Danieli, Marianna, Juliana and advisor Nemailla singing a traditional song)"
+Q[30] = "<i> That guy is your friend?</i> Izabella Neshich (UNICAMP-EMSE team student, about Thibault, after he cut his hair and become unrecognizable)"
+Q[31] = "<i> Who are you? What did you do with Thibault?</i> Juliana Canto (UNICAMP-EMSE team student, said after Thibault cut his hair and become unrecognizable)"
+Q[32] = "<i> When you are in hell, hugh satan </i> Nemailla Bonturi (UNICAMP-EMSE team advisor, explaining to Claire about the lab life infortunities)"