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Team:Grenoble/Biology/Protocols/Double
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<h1>Double mutant construction</h1> | <h1>Double mutant construction</h1> | ||
<h2>Goal</h2> | <h2>Goal</h2> | ||
| - | Obtain double gene knockout mutant strains of <i>E. coli</i> from single mutant strains. | + | Obtain double gene knockout mutant strains of <i>E. coli</i> from single mutant strains. (Keio collection)</a> |
</section> | </section> | ||
<br/> | <br/> | ||
<section> | <section> | ||
<h2>Protocol</h2> | <h2>Protocol</h2> | ||
| - | From the single mutant strains Δ<i>cyaA</i>, the first step was to remove the Kan cassette in order to create the recipient strain. The second step is using the P1 bacteriophage to transfer a Kan cassette from another bacteria to this recipient strain, in order to replace the second gene. The phage is grown on a strain containing the Kan cassette (Δ<i>araC</i> or Δ<i>envZ</i>), and the resulting phage lysate, wich contains phage DNA and bacterial DNA, is used to infect the recipient strain. Genetic recombination, catalyzed by enzymes of the recipient strain, incorporates the | + | From the single mutant strains Δ<i>cyaA</i>, the first step was to remove the Kan cassette in order to create the recipient strain <a href="#ref">[1]</a>.<br/> |
| - | <a href="http://www.ncbi.nlm.nih.gov/pubmed/18265391"> | + | <br/> |
| + | The second step is using the P1 bacteriophage to transfer a Kan cassette from another bacteria to this recipient strain <a href="#ref">[2]</a>, in order to replace the second gene. The phage is grown on a strain containing the Kan cassette (Δ<i>araC</i> or Δ<i>envZ</i>), and the resulting phage lysate, wich contains phage DNA and bacterial DNA, is used to infect the recipient strain. Genetic recombination, catalyzed by enzymes of the recipient strain, incorporates the Kan cassette into the recipient chromosome. | ||
| + | </section> | ||
| + | |||
| + | <br/> | ||
| + | <section> | ||
| + | <h1 id="ref">References</h1> | ||
| + | <ul><li><b>[1]</b> <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1681482/" target="_blank">Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.</a> | ||
| + | <li><b>[2]</b> <a href="http://www.ncbi.nlm.nih.gov/pubmed/18265391" target="_blank">E. coli genome manipulation by P1 transduction.</a></ul> | ||
</section> | </section> | ||
</div> | </div> | ||
Latest revision as of 02:41, 27 September 2012
Double mutant construction
Goal
Obtain double gene knockout mutant strains of E. coli from single mutant strains. (Keio collection)Protocol
From the single mutant strains ΔcyaA, the first step was to remove the Kan cassette in order to create the recipient strain [1].The second step is using the P1 bacteriophage to transfer a Kan cassette from another bacteria to this recipient strain [2], in order to replace the second gene. The phage is grown on a strain containing the Kan cassette (ΔaraC or ΔenvZ), and the resulting phage lysate, wich contains phage DNA and bacterial DNA, is used to infect the recipient strain. Genetic recombination, catalyzed by enzymes of the recipient strain, incorporates the Kan cassette into the recipient chromosome.
