Endy:Library-based construction

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=="Fixing" several broken inverters==
=="Fixing" several broken inverters==
We will demonstrate the utility of the [[PoPS device screening plasmid|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.
We will demonstrate the utility of the [[PoPS device screening plasmid|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.
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==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
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#*plate a portion on selecive media in order to determine the libary size.
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#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==
==Reshmaverters==

Revision as of 14:25, 11 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.

  1. generate inverter library via mutagenic PCR
  2. Digest the library and ligate into SP1.0
  3. Transform into electrocompetent CW2553/pJat8
  4. 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.
  5. Store 5 glycerols of the libary from the overnight culture
  6. Start experimental cultures with no induction and high induction
  7. 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.
  8. 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).
  9. Conduct second round of sorting to create sub-libraries that we're tested under both HIGH input and LOW input conditions.
  10. Grow up library and store 5 glycerols, as well as plate to get individual mutants.
  11. 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.

Results

TetR

Results

Mnt

penI

Results

Contact

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