Difference between revisions of "Part:BBa K644000:Experience"
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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. | 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. | 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: | Base Procedure: | ||
1. Grow strains in 1% S-Raffinose | 1. Grow strains in 1% S-Raffinose | ||
| − | 2. Transfer to 2% S-Galactose (Used 1% before like UCSF 2011, but discovered 2% was more effective | + | 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) | 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) | 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) | 4. Take time points (they vary 0,2,4,6,8,24 or 0,1.5, 3, 6, 8, 24) | ||
5. Microscopy! | 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. | * 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. | ||
| + | |||
| + | [[Image:E.Cadherin 0 24.jpg|400px|thumb|right|Figure 1: E. Cadherin in CB008DB at 24 hour time points]] | ||
| + | [[Image:CB008DB 24 UCSF2015.jpg|400px|thumb|left|Figure 2: CB008DB at 24 hour time points]] | ||
Latest revision as of 19:41, 17 September 2015
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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.
