Team:Tokyo-NoKoGen/photocontrol
From 2011.igem.org
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<p class="style61">1-3. Evaluation</p> | <p class="style61">1-3. Evaluation</p> | ||
- | <p class="style59">We expressed phototaxis device (BBa_K317028) in <em>E.coli</em> DH5α and evaluated its function. The <em>E. coli</em> was pre-cultured in 3 mL LB medium containing Chloramphenicol at | + | <p class="style59">We expressed phototaxis device (BBa_K317028) in <em>E.coli</em> DH5α and evaluated its function. The <em>E. coli</em> was pre-cultured in 3 mL LB medium containing Chloramphenicol at 37<span class="style85">˚</span>C until the OD660 reached 0.5. 1 mL of the pre-culture was removed into a tube, where all-trans retinal (f.c. 2 μM) was added. The tubes were wrapped in aluminium foil to avoid light, and were cultured by shaking at 37 <span class="style85">˚</span>C for 2 hours. 10 μL of pre-cultured solution was plotted onto semisolid medium of 0.5% agar and 2 μM all-trans retinal, and incubated overnight under light (fluorescent light) or dark (wrapped in aluminium foil) conditions. As a result, we expect to see that the size of the colony will not change under light condition because the cells will not move, but under dark condition, we expect to see that the colonies grow larger because of the induced movement.</p> |
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<p class="style59"><strong><span class="style53">2-3. Evaluation</span></strong></p> | <p class="style59"><strong><span class="style53">2-3. Evaluation</span></strong></p> | ||
- | <p class="style59">The <em>E. coli</em> cells containing Antigen43 gene was pre-cultured in 3 mL LB medium at 37 | + | <p class="style59">The <em>E. coli</em> cells containing Antigen43 gene was pre-cultured in 3 mL LB medium at 37 <span class="style85">˚</span>C for 12 hours. After adjusting its OD595, they were pre-cultured solution were suspended into 100 mL LB medium and cultured at 37 <span class="style85">˚</span>C. Its OD595 was measured every 1 hour until the OD595 reached 2.5, and 3 mL of it was put into a test tube for IPTG induction of Antigen43 expression. After 2 hours of culturing at 37 <span class="style85">˚</span>C, OD595 of the liquid culture 1cm from the surface was measured and plotted against a curve.</p> |
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<p class="style59"><strong><span class="style53">3-3. Evaluation</span></strong></p> | <p class="style59"><strong><span class="style53">3-3. Evaluation</span></strong></p> | ||
- | <p class="style59">Lysis genes with and without antiholin was evaluated to see any leakage of lysis gene expression. An <em>E. coli</em> containing an empty vector as a control, an <em>E. coli</em> containing Lysis genes with antiholin, and an <em>E. coli</em> containing Lysis genes without antiholin were cultured at 37 | + | <p class="style59">Lysis genes with and without antiholin was evaluated to see any leakage of lysis gene expression. An <em>E. coli</em> containing an empty vector as a control, an <em>E. coli</em> containing Lysis genes with antiholin, and an <em>E. coli</em> containing Lysis genes without antiholin were cultured at 37 <span class="style85">˚</span>C and OD660 was measured every 30min. to 1 hour.</p> |
<p class="style59">To find out if IPTG expression of lysis gene work, using the vectors containing holin and endolysin expressed under lac promoter, and antiholin expressed under a constitutive promoter, we transformed <em>E. coli</em> DH5α. The obtained colony pre-cultured overnight in 2 mL LB and 1 mL of it was cultured in 100 mL LB at 37℃. At OD660, IPTG (f.c. 3 mM) was added to induce the expression of Lysis genes. </p> | <p class="style59">To find out if IPTG expression of lysis gene work, using the vectors containing holin and endolysin expressed under lac promoter, and antiholin expressed under a constitutive promoter, we transformed <em>E. coli</em> DH5α. The obtained colony pre-cultured overnight in 2 mL LB and 1 mL of it was cultured in 100 mL LB at 37℃. At OD660, IPTG (f.c. 3 mM) was added to induce the expression of Lysis genes. </p> | ||
<p class="style55"> </p> | <p class="style55"> </p> |
Revision as of 11:49, 5 October 2011
Tokyo-NokoGen 2011 Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology |
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