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Team:British Columbia/Notebook/Week 12
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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. | 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. | ||
| - | Chris Keeling | + | 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. |
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Revision as of 19:35, 21 August 2011
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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.
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.
