Part:BBa_K644000:Experience
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_K644000
User Reviews
UNIQ588a01ac4a081983-partinfo-00000000-QINU UNIQ588a01ac4a081983-partinfo-00000001-QINU
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. 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. 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 difference between calcium concentrations, there was not much difference between CB008DB and E. Cadherin. Also, we sequenced the protein in pCTCON and the results showed many stop codons scattered within the data. That led us to believe that this E. Cadherin gene is not functional. 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 Calcium(CaCl2) concentrations (0, 1mM, 2mM)
4. Take time points (they vary 0,2,4,6,8,24 or 0,1.5, 3, 6, 8, 24)
5. Microscopy!
- 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.
