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Shoot for the moon. Even if you miss it,
you will land among the stars.
Les Brown
Day 7: Confirmation of ITA transformation
Assignments Due
- Go to the Presentation
Schedule
- Presenters see the guidelines for Journal
Club Presentation
- Everyone else
read listed articles and
be prepared for discussion
- Articles in OWL-Space Resources
- Tight Regulation, Modulation, and High-Level Expression
by Vectors Containing the Arabinose PBAD Promoter
(J. Bacteriology 177: 4121-4130, 1995)
- Generation of an AraC-araBAD Promoter-Regulated T7 Expression
System (Analytical Biochem. 277: 67-73, 2000)
Overview of Experiment
In preparation for lab, please read the papers (in OWL-Space
resources) about the genetic circuits we're using to test promoter
regulation. For lab days 7 and 8, we work with pET-Venus-GRX2+pTara
(in DH5alpha, resistant to kanamycin and chloramphenicol) and
pTara (in DH5alpha, resistant to chloramphenicol). This multiplasmid
circuit functions as an AND gate. Today, you design experiments
with different growth conditions to test the function of this
circuit. Also, you confirm the identity of plasmid DNA from
the ITA transformation.
- Outline of goals: How do you tune the output of the system?
- How does the system work?
- What are appropriate controls?
- How do we account for background?
- How does signal noise affect the results?
- Make a plan for Lab Day 8 (TEAM assignment)
- What to measure?
- What are good controls?
- What are potential problems?
Confirmation of Isothermal Assembly Transformation
YOU WILL HAVE TO COME IN THE DAY BEFORE LAB DAY 7 AND
PULL FOUR COLONIES (assembled vector) per team.
The colonies will be grown overnight in 3 ml LB-Amp (50 µg/ml)/Kan (20 µg/ml) at 37°C with shaking (225 rpm).
- Isolate plasmid DNA using the procedures from the first day of lab
- Digest plasmid DNA to pop-out pET-24 kan cassette
- Analyze digested DNA on 0.8% agarose gel (1 per team)
BRAINSTORM: what are other ways you could confirm that the ITA worked?
Copyright, Acknowledgements,
and Intended Use
Created by B. Beason (bbeason@rice.edu),
Rice University, 21 November 2007
Updated 2 November 2011