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Team:British Columbia/Notebook/Week 12
From 2011.igem.org
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===3-Carene synthase=== | ===3-Carene synthase=== | ||
Daisy is amplifying out the truncated synthase from the original template (which contains the EcoRI, PstI, and XbaI sites) and the mutagenized product (which does not have the cut sites). Daisy is using taq polymerase. | Daisy is amplifying out the truncated synthase from the original template (which contains the EcoRI, PstI, and XbaI sites) and the mutagenized product (which does not have the cut sites). Daisy is using taq polymerase. | ||
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| + | ===ERG20=== | ||
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| + | [[File:ubcigemgurpalatbench.jpg | frame | left | Lots of experiments. The result... an incredibly busy bench!]] | ||
Revision as of 19:20, 26 August 2011
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Contents |
Team Meeting
Progress to date: we're starting to run a lot of GC-MS in order to characterize the function of our hopefully expressed monoterpene synthases in yeast. We are also in the process of sequencing all our parts in the yeast and biobrick plasmids.
Team Social: All you can eat sukiyaki
Characterization: Limonene Synthase
Daisy is starting the expression of the Limonene synthase (which produces +limonene) in C41 DE3. Daisy has been bugging Chris Keeling a lot about the expression conditions. It turns out that Daisy needs to do an in vitro assay for limonene synthase (adding GPP + purified enzyme). The only problem is there is no tag for a purification step. So it will most likely be the limonene synthase + a whole bunch of other enzymes + GPP (substrate needed to convert to limonene).
This is potentially problematic since it is much harder to do an in vitro assay without a purification step. Daisy originally planned to do a 500 mL expression, as suggested by Bohlmann Lab members, which is what they usually do. However, Daisy is now going to do a 2 L or 3 L expression. It will be better to have more enzyme rather than less for the in vitro assay.
Daisy has a lot of questions to ask Chris Keeling about the expression conditions. However, Chris Keeling went on vacation so Daisy will need to ask other lab members.
Daisy has now expressed the C41 DE3 cells in terrific broth with an overnight expression. Daisy has spun down the cells and is going to run some of the cell lysate on an SDS PAGE (pre-induction and post induction). Daisy will also be running some of Vicki and Jacob's sample on the SDS PAGE.
3-Carene synthase
Daisy is amplifying out the truncated synthase from the original template (which contains the EcoRI, PstI, and XbaI sites) and the mutagenized product (which does not have the cut sites). Daisy is using taq polymerase.
ERG20
