Endy:Library-based construction
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==Procedure== | ==Procedure== | ||
This should be moved to its own page and fleshed out as we work through it in the lab. | This should be moved to its own page and fleshed out as we work through it in the lab. | ||
| - | #generate inverter library via mutagenic PCR | + | #generate inverter library via [[Endy:Mutagenic PCR|mutagenic PCR]] |
| - | #Digest the library and ligate into SP1.0 | + | #Digest the library and ligate into [[Endy:Screening plasmid|SP1.0]] |
#Transform into electrocompetent CW2553/pJat8 | #Transform into electrocompetent CW2553/pJat8 | ||
#Grow for one hour without antibiotic (1ml), then move into a higher volume (50ml) of selective media | #Grow for one hour without antibiotic (1ml), then move into a higher volume (50ml) of selective media | ||
Revision as of 13:57, 13 July 2006
This is a work in progress, bear with us.
Contents |
"Fixing" several broken inverters
We will demonstrate the utility of the screening plasmid for fixing "broken" inverters via directed evolution. Preliminary results with the tetR-based inverter <bbpart>Q04400</bbpart> showed early success of this method.
Procedure
This should be moved to its own page and fleshed out as we work through it in the lab.
- generate inverter library via mutagenic PCR
- Digest the library and ligate into SP1.0
- Transform into electrocompetent CW2553/pJat8
- Grow for one hour without antibiotic (1ml), then move into a higher volume (50ml) of selective media
- plate a portion on selecive media in order to determine the libary size.
- Store 5 glycerols of the libary from the overnight culture
- Start experimental cultures with no induction and high induction
- Sort cells with LOW in/HIGH out and HIGH in/LOW out into sub-libraries.
- Be sure to also collect analytical samples of the populations after sorting, so that we have data of what the libraries looked like.
- Grow up the sub-libraries under the reverse condition (and the same condition).
- The reason for growing under the same conditions is to see how effective the sorting was (control, basically).
- Conduct second round of sorting to create sub-libraries that we're tested under both HIGH input and LOW input conditions.
- Grow up library and store 5 glycerols, as well as plate to get individual mutants.
- Test individual mutants, if necesary can repeat the rounds of screening to tighten up the library.
Reshmaverters
We are working with Reshma Shetty to test her new inverter designs.
- Knight:Evolving Reshmaverters - project page.
Results
TetR
Results
Need to port the results described here.
