Team:Marburg:Project:Notebook:May
From 2014.igem.org
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</table> | </table> | ||
- | <p>The isolated plasmids were | + | <p>The isolated plasmids were digested with <i>Nco</i>I for 1h 20 min and further analyzed on a 1% agarose gel.</p> |
</div> | </div> | ||
<div class="results"> | <div class="results"> | ||
<h3>Results</h3> | <h3>Results</h3> | ||
<img src="https://static.igem.org/mediawiki/2014/5/5e/MR_2014-05-06_NcoI.PNG" width="30%" /> | <img src="https://static.igem.org/mediawiki/2014/5/5e/MR_2014-05-06_NcoI.PNG" width="30%" /> | ||
- | <p>Restriction of the isolated pMa12 with <i>Nco</i>I; the lanes contain the | + | <p>Restriction of the isolated pMa12 with <i>Nco</i>I; the lanes contain the digested pMa12 in the order of the picked clones 1 - 15; clone 9 was negative; the lanes between the restrictions and the marker contain the isolated pMAD but it appears to be empty.<br /> |
The most clones contained a plasmid that has only one <i>Nco</i>I restriction site. The expected size of the pMa12 should be around 6.6 kb. All fragments are of the expected size.</p> | The most clones contained a plasmid that has only one <i>Nco</i>I restriction site. The expected size of the pMa12 should be around 6.6 kb. All fragments are of the expected size.</p> | ||
</div> | </div> | ||
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</h2> | </h2> | ||
<fieldset class="exp15"> | <fieldset class="exp15"> | ||
- | <legend><a name="exp15.22">15.22 Restriction of 3 | + | <legend><a name="exp15.22">15.22 Restriction of 3 isolated plasmids (6.5.14) with <i>Nco</i>I and <i>Bam</i>HI</a></legend> |
<div class="aim"> | <div class="aim"> | ||
<p>Aim: Test whether the isolated plasmid contains the 2 kb insert (hag-flank construct).</p> | <p>Aim: Test whether the isolated plasmid contains the 2 kb insert (hag-flank construct).</p> | ||
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</fieldset> | </fieldset> | ||
<fieldset class="exp13"> | <fieldset class="exp13"> | ||
- | <legend><a name="exp13.43">13.43 Ligation of the annealed oligos into | + | <legend><a name="exp13.43">13.43 Ligation of the annealed oligos into digested piGEM001</a></legend> |
<div class="aim"> | <div class="aim"> | ||
<p>Aim: Create plasmid with the corresponding promoter sequences for Ag/Cu</p> | <p>Aim: Create plasmid with the corresponding promoter sequences for Ag/Cu</p> | ||
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</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>The plasmid pMAD was isolated and | + | <p>The plasmid pMAD was isolated and digested with the restriction enzymes <i>Bam</i>HI and <i>Nco</i>I to create fitting ends for the ligation with the digested PCR-fragment.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
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</fieldset> | </fieldset> | ||
<fieldset class="exp15"> | <fieldset class="exp15"> | ||
- | <legend><a name="exp15.34">15.34 Ligation of | + | <legend><a name="exp15.34">15.34 Ligation of digested pMAD with the digested hag/flank-Construct and trafo</a></legend> |
<div class="aim"> | <div class="aim"> | ||
<p>Aim: link the fragment with the vector and trafo of DH5α</p> | <p>Aim: link the fragment with the vector and trafo of DH5α</p> | ||
</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>The ligation of pMAD and the | + | <p>The ligation of pMAD and the digested PCR-fragment was done according to the previous mentioned ligation calculator.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
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</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>The plasmid pMAD from 13.05.14 (ca. 25µl) and | + | <p>The plasmid pMAD from 13.05.14 (ca. 25µl) and digested with the restriction enzymes <i>Bam</i>HI and <i>Nco</i>I to create fitting ends for the ligation with the digested PCR-fragment. The digest was performed in 2 Eppis with 12,5µl plasmid each.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
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</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>The ligation of pMAD and the | + | <p>The ligation of pMAD and the digested PCR-fragment was done according to the previous ligation calculator.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
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</fieldset> | </fieldset> | ||
<fieldset class="exp17"> | <fieldset class="exp17"> | ||
- | <legend><a name="exp17.3">17.3 First Plate Reader Measurement with <i>Bacillus subtilis</i> wildtype and strain 186 | + | <legend><a name="exp17.3">17.3 First Plate Reader Measurement with <i>Bacillus subtilis</i> wildtype and strain 186 containing a <i>gfp</i></a></legend> |
<div class="aim"> | <div class="aim"> | ||
<p>Aim: Test for differences between the two strains and first check if experiment works at all</p> | <p>Aim: Test for differences between the two strains and first check if experiment works at all</p> | ||
</div> | </div> | ||
+ | <br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/3/3b/MR_20140520_MTA_growth_Bsubtilis_wt_gfp_MM_LB.jpg" width="40%" /> | ||
+ | <br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/0/09/MR_20140520_MTA_fluorescence_bsubtilis_wt_gfp_MM_xyl_LB.jpg" width="40%" /> | ||
+ | <br /> | ||
+ | <p>Both strains grew best in LB medium like expected, a clearly better growth was shown by the wildtype in the minimal medium compared to the <i>gfp</i> bearing strain.</p> | ||
+ | <p>The fluorescence is shown in dependence of the culture growth in order to relativise the fluorescence signal. Like expected, the wt exhibited no fluorescence in both media. The fluorescence of the <i>gfp</i> strain was higher in the minimal medium with xylose, than without xylose.</p> | ||
</fieldset> | </fieldset> | ||
</div> | </div> | ||
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<fieldset class="exp15"> | <fieldset class="exp15"> | ||
- | <legend><a name="exp15.45">15.45 Gibson Assembly of | + | <legend><a name="exp15.45">15.45 Gibson Assembly of digested pMad-fla and cup 1 and Transformation into <em>E.Coli</em> XL1-Blue</a></legend> |
<div class="aim"> | <div class="aim"> | ||
<p>Aim: create a flagellin that contains the metallothinein cup1</p> | <p>Aim: create a flagellin that contains the metallothinein cup1</p> | ||
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<legend><a name="exp17.6">17.6 MTA Bacillus Nose Ag </a></legend> | <legend><a name="exp17.6">17.6 MTA Bacillus Nose Ag </a></legend> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p> | + | <p>Transformation from 20.5.14: one colony on the Ag Nose plate<br /> |
- | Inoculation of shaking culture in the morning together with the wildtype and the gfp strain | + | Inoculation of shaking culture in the morning together with the wildtype and the <i>gfp</i> strain in LB liquid without antibiotics</p> |
- | <p> | + | <p>Inoculate 96 well plate in the afternoon 15:00</p> |
<p>Prepare Cu and Ag solutions to test different concentrations in MTA and see if the Ag nose responds to Ag and also if it is specific for Ag and does not respond to Cu</p> | <p>Prepare Cu and Ag solutions to test different concentrations in MTA and see if the Ag nose responds to Ag and also if it is specific for Ag and does not respond to Cu</p> | ||
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<p>Ag and Cu in stock solution of 10 mM have been prepared<br /> | <p>Ag and Cu in stock solution of 10 mM have been prepared<br /> | ||
Added (Concentration): 11 mM, 100µM and 10µM<br /> | Added (Concentration): 11 mM, 100µM and 10µM<br /> | ||
- | Cells: Ag Nose mutant, wildtype (AG GM) and gfp | + | Cells: Ag Nose mutant, wildtype (AG GM) and <i>gfp</i></p> |
+ | <br /> | ||
+ | <p>The MTA showed no significant increase of fluorescence. The bacteria had difficulties in growth, thus it seemed that the metal ion concentrations were too high.</p> | ||
</div> | </div> | ||
</fieldset> | </fieldset> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
- | <th scope="row"><strong>Template | + | <th scope="row"><strong>Template ~10µg</strong></th> |
<td>15</td> | <td>15</td> | ||
<td>20</td> | <td>20</td> | ||
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</table> | </table> | ||
<p>The restriction was carried out at 37°C for 1h.<br /> | <p>The restriction was carried out at 37°C for 1h.<br /> | ||
- | The restriction was cleaned with a DNA Gel Ex Kit (c = 40 ng/µl). 4µl (160 ng) of this | + | The restriction was cleaned with a DNA Gel Ex Kit (c = 40 ng/µl). 4µl (160 ng) of this digested plasmid were mixed with 1 µl of the cup1-1 fragment (9.6 ng/µl). These 5 µl were inserted into the prepared 15 µl Gibson-mix. The reaction was incubated at RT for 1 min and then at 50°C for 1 hour. An extra Gibson-reaction was carried out as a control with just the linearized pMAD without the <i>cup</i>1-1.<br /> |
<em>E. coli</em> DH5α cells were transformed with the Gibson reactions and plated out on LB-Amp. They were incubated at 37°C over night.</p> | <em>E. coli</em> DH5α cells were transformed with the Gibson reactions and plated out on LB-Amp. They were incubated at 37°C over night.</p> | ||
</div> | </div> | ||
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</table> | </table> | ||
<p>The restriction was incubated at 37°C for 1 h.</p> | <p>The restriction was incubated at 37°C for 1 h.</p> | ||
- | <p>The restriction mix was purified with the Gel Ex Kit. The yield was too low (c = 3.1 ng/µl) to use it for a Gibson-Assembly, so the rest of the pET24d-fla was | + | <p>The restriction mix was purified with the Gel Ex Kit. The yield was too low (c = 3.1 ng/µl) to use it for a Gibson-Assembly, so the rest of the pET24d-fla was digested with <i>Spe</i>I.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
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</table> | </table> | ||
<p>The restriction was incubated at 37°C for 1h.</p> | <p>The restriction was incubated at 37°C for 1h.</p> | ||
- | <p>The restriction mix was purified with the Gel Ex Kit (c = 20.1 ng/µl), which was enough for the Gibson-Assembly. 4µl (80 ng) of this | + | <p>The restriction mix was purified with the Gel Ex Kit (c = 20.1 ng/µl), which was enough for the Gibson-Assembly. 4µl (80 ng) of this digested plasmid were mixed with 1 µl of the cup1-1 fragment (9.6 ng/µl). These 5 µl were inserted into the prepared 15µl Gibson-mix. The reaction was incubated at RT for 1 min and then at 50°C for 1 h. An extra Gibson-reaction was carried out as a control with just the linearized pET24d-fla without the <i>cup</i>1-1.</p> |
<p>Competent <em>E. coli</em> XLI-Blue were transformed with the whole Gibson-reactions. They were plated on LB-Kan an incubated at 37°C overnight.</p> | <p>Competent <em>E. coli</em> XLI-Blue were transformed with the whole Gibson-reactions. They were plated on LB-Kan an incubated at 37°C overnight.</p> | ||
</div> | </div> | ||
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<div class="exp-content"> | <div class="exp-content"> | ||
<p>The positive clone 3 from the colony PCR (28.05.14) was used to inoculate 6 ml LB-Amp for a miniprep in order to receive the plasmid for transforming <em>Bacillus subtilis</em>.<br /> | <p>The positive clone 3 from the colony PCR (28.05.14) was used to inoculate 6 ml LB-Amp for a miniprep in order to receive the plasmid for transforming <em>Bacillus subtilis</em>.<br /> | ||
- | Incubation at room temperature over | + | Incubation at room temperature over two days.</p> |
</div> | </div> | ||
</fieldset> | </fieldset> |
Latest revision as of 19:28, 16 October 2014
Notebook: May