Part:BBa_K197018:Experience
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Applications of BBa_K197018
The 2011 UCSF iGEM Team Characterized this part by expressing it on the surface of yeast. You can see more information on our wiki page: [http://2011.igem.org/Team:UCSF/MGFP5 Mgfp-5 Characterization]
Our results show conclusive evidence that mgfp-5-expressing yeast “cluster”, form clumps of cells that grow on and around on another, more often and to a much larger degree than non-mgfp-5-expressing yeast. We hope that this could be used to create a biomaterial or a bioadhesive that could be natural and biodegradable.
Figure 1: Control, non-induced mgfp- 5 yeast. Dispersed, no major clustering.
Figure 2: Experiment, induced mgfp- 5 yeast. 3-D cluster of yeast cells growing around and on top of other cells.
UCSF iGEM 2015
Eleanor Amidei
Our project this year included a clustering element, so we looked at Mgfp-5, Hwp1, and E. Cadherin.
We began by testing them by using the parameters UCSF 2011 laid out, but instead of using artificial seawater, we used NaCl, which may serve as a possible explanation for our results. What we found was that everything clustered after 24 hours, indicating that maybe it was more about population density than actual clustering. We tested the clustering protein at different population densities to test this theory. Higher densities clustered more easily than lower density cultures.
At all of our time points earlier than 24 hours, the control, CB008DB did not cluster, while mgfp5 did.
We then transformed them into our own strain of yeast, CB008BD, a knockout strain without Bar 1. We again followed similar parameters to UCSF 2011 but included a CB008DB strain as a control. Again, we found that after 24 hours, everything began to cluster. Though there was a slight difference between NaCl concentrations, there was not much difference between CB008DB and Mgfp5 overall.
Mgfp5 seemed to clump the most after 24 hours, though E. Cadherin seemed more effective up to the 8 hour time point. We placed this gene downstream of a transcription factor inducible promoter, which yielded increased cluster size
Base Procedure:
1. Grow strains in 1% S-Raffinose
2. Transfer to 2% S-Galactose (Used 1% before like UCSF 2011, but after doing some research, discovered 2% was more effective.)
2.5 Dilute to desired OD (LD - .001 HD - .1, our project experiment - 0.015)
3. Induce with different NaCl concentrations (0, 100mM, 200mM)
4. Take time points (they vary 0,2,4,6,8,24 or 0,1.5, 3, 6, 8, 24)
5. Microscopy!
- 400mM NaCl seemed to decrease clumping*
- We followed the guidelines set by UCSF 2011 but altered them to function better. The concentrations/procedure listed above are what we used generally throughout our preliminary testing phase, but it varied a bit from experiment to experiment.
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