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Team:Grenoble/Biology/Protocols/AND test
From 2012.igem.org
(Difference between revisions)
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<section> | <section> | ||
<h1>"AND" gate test</h1> control of pBAD expression by cAMP and arabinose. | <h1>"AND" gate test</h1> control of pBAD expression by cAMP and arabinose. | ||
| + | </section> | ||
| + | <section> | ||
<h2>Goal</h2> | <h2>Goal</h2> | ||
Measure the dependence of the activity of the pBAD promoter on the concentrations of cAMP and arabinose. | Measure the dependence of the activity of the pBAD promoter on the concentrations of cAMP and arabinose. | ||
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<h2>Procedure</h2> | <h2>Procedure</h2> | ||
<h3>Outline</h3> | <h3>Outline</h3> | ||
| - | The day before: Grow BW25113 cya<span class="exposant">-</span> containing the pAra/Bad_RBS_GFP plasmid of Alon in LB + Kanamycin. Leave on the shaker at 37°C until the next day morning. | + | The day before: Grow BW25113 cya<span class="exposant">-</span> containing the pAra/Bad_RBS_GFP plasmid of Alon in LB + Kanamycin. Leave on the shaker at 37°C until the next day morning.<br/> |
Measure expression in different concentrations of arabinose and cAMP in M9 added with glucose, acetate or glycerol. | Measure expression in different concentrations of arabinose and cAMP in M9 added with glucose, acetate or glycerol. | ||
</section> | </section> | ||
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<h2>Protocol</h2> | <h2>Protocol</h2> | ||
<h3>Outline</h3> | <h3>Outline</h3> | ||
| - | Add 10 microliters of an appropriate mix of cAMP and arabinose to each well of a 96-well microplate (see plate layout below). Add 130 microliters of M9 into each well. The medium is prepared fresh, but not filtered. Then, add 10 microliters of the o/n culture and read in Fusion at 37°C. | + | Add 10 microliters of an appropriate mix of cAMP and arabinose to each well of a 96-well microplate (see plate layout below). |
| - | cAMP: 0 to 3.2 mM: 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 mM (dilution by 2-fold) | + | Add 130 microliters of M9 into each well. The medium is prepared fresh, but not filtered. |
| - | arabinose: 0 to 0.1%: 0.1, 0.02, 0.004, 0.0008, 0.00016, 0 % (5-fold dilutions) | + | Then, add 10 microliters of the o/n culture and read in Fusion at 37°C.<br/> |
| + | cAMP: 0 to 3.2 mM: 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 mM (dilution by 2-fold)<br/> | ||
| + | arabinose: 0 to 0.1%: 0.1, 0.02, 0.004, 0.0008, 0.00016, 0 % (5-fold dilutions)<br/> | ||
</section> | </section> | ||
Revision as of 14:14, 22 August 2012
"AND" gate test
control of pBAD expression by cAMP and arabinose.Goal
Measure the dependence of the activity of the pBAD promoter on the concentrations of cAMP and arabinose.Procedure
Outline
The day before: Grow BW25113 cya- containing the pAra/Bad_RBS_GFP plasmid of Alon in LB + Kanamycin. Leave on the shaker at 37°C until the next day morning.Measure expression in different concentrations of arabinose and cAMP in M9 added with glucose, acetate or glycerol.
Protocol
Outline
Add 10 microliters of an appropriate mix of cAMP and arabinose to each well of a 96-well microplate (see plate layout below). Add 130 microliters of M9 into each well. The medium is prepared fresh, but not filtered. Then, add 10 microliters of the o/n culture and read in Fusion at 37°C.cAMP: 0 to 3.2 mM: 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 mM (dilution by 2-fold)
arabinose: 0 to 0.1%: 0.1, 0.02, 0.004, 0.0008, 0.00016, 0 % (5-fold dilutions)
