Team:UNICAMP-EMSE Brazil/Methodology

From 2011.igem.org



Contents

Creating a great WARRIOR!

In order to create our “Jedi coli” to fight the STRESS WARS, we trained padawcolis to use their power to sense Nitric Oxide (NO) or Adrenaline and produce IL-10 and IL-12, respectively, to defend or bodyConfederation host against inVader bacteria. By the end of the harsh Jedi training, which included hours of cutting and ligation, cloning and transformation, we aimed to develop the following gene constructions:


UNICAMP EMSE secretion METHODOLOGY1.jpg

Work plan:
Our work was divided in three main steps:

  1. Biobrick assembly;
  2. Suit the biobrick for shipment (remove them from the original plasmid and insert them on pSB1C3 plasmid);
  3. Devices construction and testing.

First step: Biobricks Assembly

Part descriptions

For the reasons explained in the safety proposals, we synthesized the following gene sequences according to assembling standard 23 and in the format of transcriptional units [with RBS ([http://partsregistry.org/Part:BBa_B0030 B0030]) and stop codon, when protein coding:

UNICAMP EMSE flhdc.jpg
flhDC promoter, part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K554001 BBa_K554001]


UNICAMP EMSE soxs.jpg
SoxS promoter, part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K554000 BBa_K554000]


UNICAMP EMSE soxr.jpg
RBS+SoxR gene, part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K554003 BBa_K554003]


UNICAMP EMSE il10.jpg
RBS+IL-10 gene, part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K554004 BBa_K554004]


UNICAMP EMSE il12.jpg
RBS+IL-12 gene, part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K554005 BBa_K554005]


UNICAMP EMSE HlyB.jpg
RBS+Hemolysin B(HlyB) gene, part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K554007 BBa_K554007]


UNICAMP EMSE hlyD.jpg
RBS+Hemolysin D(HlyD) gene, part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K554008 BBa_K554008]


UNICAMP EMSE TolC.jpg
RBS+TolC gene, part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K554009 BBa_K554009]


UNICAMP EMSE qsecqseb.jpg
RBS+QseB gene+QseC gene, part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K554006 BBa_K554006]


UNICAMP EMSE hlyA.jpg
RBS+HlyA coding secretion signal sequence, part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K554002 BBa_K554002]


In our project, we also used the following biobricks from the 2011´s biobrick collection distribution sent by the iGEM headquarters:


UNICAMP EMSE const.jpg
Constitutive promoter, part [http://partsregistry.org/Part:BBa_J23119 BBa_J23119]


UNICAMP EMSE gfp.jpg
GFP gene, part [http://partsregistry.org/Part:BBa_E0040 BBa_E0040]


UNICAMP EMSE term.jpg
Double terminator, part [http://partsregistry.org/Part:BBa_B0014 BBa_B0014]


UNICAMP EMSE gfp.jpg
RBS.1 (strong) , part [http://partsregistry.org/Part:BBa_B0030 BBa_E0030]


Description of the assembly process:

  1. NO and Adrenaline sensors:
  • The first step was to ligate both SoxR and QseB/QseC transcriptional units with the terminator, followed by the ligation of these constructs to the constitutive promoter.
  1. IL-10, IL-12 and GFP expressing units:
  • Terminator sequence was ligated in the Hemolysin A sequence, followed by the fusion of this construction with the IL-10, IL-12 or GFP transcriptional units (GFP one was first coupled with RBS sequence). The final biobrick was obtained after the coupling of interleukin or GFP transcription units (+ Hemolysin A + Terminator) with the respective promoters.
  1. Secretion systems:
  • The first round of ligation was accomplished by coupling the Terminator with the TolC transcriptional unit. The subsequent ligation round comprised the ligation of HlyD in the TolC+Terminator construct, while HlyB was ligated in the promoters. The last assembling step was accomplished by coupling the SoxS or flHDC promoter+HlyB with the previously assembled construction HlyD+TolC+T.

Second step: Suit the biobricks for shipment

This step was accomplished by the digestion of our biobricks with EcoRI and PstI in their initial plasmid (synthetic or iGEM backbones) and ligate them in the linearized pSB1C3 backbone.

Third step: Construction of the devices and testing

In order to ease the functional tests intended to be performed in our assembled devices, both sensor and expressing system were coupled in the same vector, resulting in the following assembled devices:

  1. Testing device to validate SoxS and sensor system:
UNICAMP EMSE method2.jpg



  1. Testing device to validate flHDC and sensor system:
UNICAMP EMSE method3.jpg



  1. To test the secretion system we made a double transformation with these devices in two different plasmids with different antibiotics resistance:
UNICAMP EMSE method4.jpg



We were able to successfully assembly those constructs but unfortunately, we did not have time to test the following assembly:

UNICAMP EMSE method5.jpg

Results

Check our device testing experiments at Results page and the day-to-day work done to built the devices at our Notebook page.