Team:Concordia/Results

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  • Lactococcus Lactis
  • Scaffold
  • Food Intolerances
  • Prototype Pathway
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  • Results of Testing
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Results of Testing

Assay Methods

Beta-Galactosidase Assay

• Chromogenic substrate ONPG
• Monitoring appearance of yellow colour over time

Sucrase Assay

• Chromogenic substrate
• Monitoring appearance of colour over time

ADH and ALD Assay

• Measure the production of NADH over time
• Rate of change at 340nm
• Calculate Kcat based on Vmax and enzyme concentration

Chromoprotein Test: aeBlue and eforRed

The first step in testing our extracellular scaffold is attempting to attach a reporter protein to account for its presence and stability. For this purpose, we are using the chromoproteins submitted by the iGEM Uppsala University 2012 team. We chose the two chromoproteins aeBlue and eforRed (respectively BBa_K864401 and BBa_K592012 on the iGEM registry) and fused them to the dockerin domains from Clostridium thermocellum cellulosome to allow them to bind to the scaffold’s cohesins.

After incubation of the chromoprotein-dockerin fusions and the scaffold-expressing variant of Lactococcus lactis, the cells are washed by several rounds of centrifugation and resuspension so that unbound proteins are washed away. After washing, if the colour associated with the chromoproteins remains in the cell portion, the protein-fusions have successfully attached to the scaffold.

This proof of concept allows us to confirm the expression, secretion, and attachment of the scaffold; as well as the integrity of the cohesin-dockerin interaction under standard conditions.

Project Achievements

Successes

• Transformed and induced scaffold into L. lactis
• Induced, purified and assayed LacZ fused with dockerins

Failure to:

• Induce chromoproteins in E. coli (no colour was visible)
• Purify SacC and pathway enzymes (assay did not work)
• It is possible, that dockerin fusion changes our protein structure and thus caused no colour to appear in chromoproteins and the failure to assay enzymes
• Bind enzyme fusions to scaffold on L. lactis
• Assay enzyme activity of enzymes docked onto scaffold