Team:Aalto-Helsinki/Achievements

<!DOCTYPE html> Aalto-Helsinki

Achievements

Main results


Detection


Created devices that produce YFP in response to oxidative stress

Based on our studies of inducing stress promoters with hydrogen peroxide, it seemed that the CCP1 and TSA1 promoters were the most promising ones. In both of these promoters, increasing H 2 O 2 concentrations seem to lead to stronger activation of promoters and thus to more intensive fluorescence signals, as seen from results obtained from microplate reader results where fluorescence is normalized to cell density (Figure 1). The functionality of the promoters was confirmed with fluorescence measurements performed with flow cytometry (Figure 2).



Figure 1. Effect of different H 2 O 2 concentrations on fluorescence as a function of OD in Venus under CCP1 promoter (left) and Venus under TSA1 promoter (right) in SS328-leu strain n=3.


Figure 2. Median fluorescence produced by the different promoters after 2 h induction (left) and fluorescence values normalized to the uninduced values (right) as a function of H 2 O 2 induction concentration.


We managed to produce a phenotype change in yeast by replacing QDR2 transporter

Using our foam-based visual catalase assay, we managed to prove a phenotype change after replacing the original QDR2 transporter in the S. cerevisiae strain SS328-leu genome with the corresponding transporter from strain VL3. This can be seen in increased catalase activity levels compared to the non-modified strain after incubation in different concentrations of microcystin (figure 3). Despite the increase of catalase activity base level, the assay didn’t give definitive proof of the functionality of the integrated transporter, as the observed response to microcystin concentrations in SS328-leu with QDR2 transporter was different than in VL3. However, this response might also be partially obscured by the large margin of error in our experiment.



Figure 3. Relative catalase activity in different yeast strains when exposed to different concentrations of microcystin.


Based on the phenotype change, we assumed that the replaced transporter was functional. However, when we attempted to induce fluorescence of our stress promoters in the strain with the replaced transporter, we didn’t obtain any fluorescent signal compared to uninduced values (figure 4). In this experiment, varying microcystin concentrations did not significantly affect any of our promoters, which we concluded to be due to the inability of the transporter to import the toxin into the cell.



Figure 4. Results of flow cytometry analysis. Effect of microcystin on yellow fluorescence production under different stress promoters in SS328-leu with and without (w/o) QDR2 transporter from VL3.


Modelling!!



Degradation


We pinpointed the localization of two MlrA constructs in yeast

Our results show that our constructs, MlrA and MlrA with mating factor alpha, both localized in the fraction comprising of inclusion bodies, plasma membrane and cell wall (figure 5, fractions P2). MlrA was detected also from fraction included only cell wall and plasma membrane (P3 fraction), indicating that the protein is localized in the plasma membrane. The absence of MlrA with the mating factor alpha from the S3 and P3 samples is most likely due to technical errors in the experiment, and, based on this, we can’t conclude if the protein is in the plasma membrane or in the inclusion bodies.



Figure 5: Localization of MlrA (MlrAY) and MlrA with mating factor alpha (MlrAYa). LAD - molecular weight standard, NC - S. cerevisiae SS328-leu without MlrA plasmid as negative control, S1 - secreted protein, S2 - soluble protein and inner membranes, P2 - inclusion bodies, plasma membrane and cell wall, S3 - refolded protein, P3 - plasma membrane and cell wall, S4 - soluble protein, P4 - inner membranes.


We proved that our enzymes are active

Based on our final enzyme activity assay, both MlrA and MlrA with mating factor alpha have activity. The earlier assay with non-diluted enzyme had shown that either there is high activity or that there is no activity at all, but with this final activity assay we managed to prove that the enzymes degrade MC-LR. The results (figure 6) show that the three different dilution degrade the MC-LR with different rates, 1:10 dilution having the highest activity and 1:1000 having the lowest. Based on the graphs it seems that the plain MlrA has a bit higher activity than MlrA with the mating factor alpha.



Figure 6: Enzyme activity of MlrA (left graph) and MlrA with mating factor alpha (right graph). The graphs show the degradation of MC-LR with three different dilutions. MlrAY - MlrA, MlrAYa - MlrA with mating factor alpha.


Based on the localization we were able to say that MlrA resides in the plasma membrane, but we were unable to conclude about the localization of MlrA with the mating factor alpha. Anyhow, after the enzyme activity assay, we are able to conclude that since both constructs were localized in the P2 fraction (and MlrA in the P3 fraction) and since both had activity, it is most likely that they both reside in the plasma membrane in an active form.



“ Cyanobacteria range in size from 0.5 to 60 micrometers in diameter which represents one of the largest prokaryotic organism. ”



Discussion





“ Cyanobacteria range in size from 0.5 to 60 micrometers in diameter which represents one of the largest prokaryotic organism. ”



Parts



Sesame snaps tootsie roll macaroon sesame snaps gingerbread jelly beans muffin. Bear claw gummi bears macaroon. Toffee chocolate ice cream. Jelly-o dessert sweet roll fruitcake tart. Sweet sesame snaps chocolate cake. Bonbon dragée gingerbread. Powder chocolate bar topping powder jujubes biscuit sugar plum. Candy dessert liquorice powder. Candy canes chocolate cake gingerbread bear claw pudding. Chupa chups croissant pie lemon drops sweet roll cheesecake. Lemon drops dessert bonbon bonbon wafer. Bear claw biscuit oat cake dessert cookie cheesecake pie liquorice macaroon. Chupa chups chupa chups muffin dragée lemon drops cake tootsie roll. Soufflé chocolate dragée oat cake pastry lemon drops cookie.Gummi bears ice cream tiramisu macaroon. Tiramisu tart powder cookie sweet roll jelly dessert dessert sweet roll. Wafer danish biscuit wafer icing jelly-o wafer.



“ Cyanobacteria range in size from 0.5 to 60 micrometers in diameter which represents one of the largest prokaryotic organism. ”



Medal Criteria



Bronze Medal

  • ✔ Register for iGEM, have a great summer, and attend the Giant Jamboree.
  • ✔ Meet all deliverables on the Requirements page.
  • ✔ Create a page on your team wiki with clear attribution of each aspect of your project. See our attributions page.
  • ✔ Document at least one new standard BioBrick Part or Device central to your project and submit this part to the iGEM Registry.



Silver Medal

  • ✔ Experimentally validate that at least one new BioBrick Part or Device of your own design and construction works as expected. Submit this new part to the iGEM Parts Registry. This working part must be different from the part documented in bronze medal criterion #4.
  • ✔ Convince the judges you have helped any registered iGEM team in a significant way. Read more about this on our community page.
  • ✔ iGEM projects involve important questions beyond the lab bench, for example relating to (but not limited to) ethics, sustainability, social justice, safety, security, and intellectual property rights. Demonstrate how your team has identified, investigated, and addressed one or more of these issues in the context of your project. More information can be found on our Human Practices page.



Gold Medal

At least two criteria must be met:

  • ✔ Expand on your silver medal activity by demonstrating how you have integrated the investigated issues into the design and/or execution of your project.
  • ✔ Improve the function OR characterization of an existing BioBrick Part or Device and enter this information in the Registry.
  • ✔ Demonstrate a functional proof of concept of your project. Your proof of concept must consist of a BioBrick device; a single BioBrick part cannot constitute a proof of concept.
  • ✘ Show your project working under real-world conditions. To achieve this criterion, you should demonstrate your whole system, or a functional proof of concept working under simulated conditions in the lab.