http://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_7&feed=atom&action=historyTeam:Cambridge/Diary/Week 7 - Revision history2024-03-29T14:52:18ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_7&diff=292986&oldid=prevEmmyft at 00:15, 27 October 20122012-10-27T00:15:41Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>More transformations of the Gibson products (Gibson control, fluorescent construct) by Emmy and Paul, along with the YFP donated by Jim H. No growth yet for the transformation done yesterday, so we'll leave it again overnight, along with the others.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>More transformations of the Gibson products (Gibson control, fluorescent construct) by Emmy and Paul, along with the YFP donated by Jim H. No growth yet for the transformation done yesterday, so we'll leave it again overnight, along with the others.</div></td></tr>
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</table>Emmyfthttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_7&diff=286504&oldid=prevOlijme at 16:42, 26 October 20122012-10-26T16:42:03Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:Cambridge/Diary/Week 13|13]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:Cambridge/Diary/Week 13|13]]</div></td></tr>
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</table>Olijmehttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_7&diff=111653&oldid=prevOlijme at 12:18, 13 September 20122012-09-13T12:18:54Z<p></p>
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</table>Olijmehttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_7&diff=87605&oldid=prevEmmyft at 08:35, 30 August 20122012-08-30T08:35:31Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Monday===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Monday===</div></td></tr>
</table>Emmyfthttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_7&diff=74558&oldid=prevEmmyft: /* Wednesday */2012-08-19T16:31:52Z<p><span class="autocomment">Wednesday</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Wednesday===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Wednesday===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We are all at that stage where our various pieces of DNA need putting into cells. To that end, we began transformations, and lots of them. We started using the electroporation device for the first time today, possibly making lots of cells with plasmids shoved through their walls. Or else we've just made a load of dead bacteria. Only a night in the incubator will tell.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We are all at that stage where our various pieces of DNA need putting into cells. To that end, we began transformations, and lots of them. We started using the electroporation device for the first time today, possibly making lots of cells with plasmids shoved through their walls. Or else we've just made a load of dead bacteria. Only a night in the incubator will tell.</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Desperate to find out the reasons behind the failure to Gibson our fluorescent contruct, Emmy did a restriction digest on the Gibson product of the fluorescent components- and there were no bands. She suspects that there wasn't enough DNA in the sample to be shown in the gel. Looks like this diagnostic test can only be performed after we successfully transform E.coli with the construct, grow them up and miniprep the plasmid. With our lack of functioning competent cells looks like it's not going to happen until earliest next week.</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We also began thinking about what we're going to show at the meet up in a week. We've been invited down to the Google campus in London by the UEA team to discuss (and more importantly, present) our projects. Paul and Emmy therefore began creating a poster for it, and Oli and Tom began talking about how to order our presentation.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We also began thinking about what we're going to show at the meet up in a week. We've been invited down to the Google campus in London by the UEA team to discuss (and more importantly, present) our projects. Paul and Emmy therefore began creating a poster for it, and Oli and Tom began talking about how to order our presentation.</div></td></tr>
</table>Emmyfthttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_7&diff=74546&oldid=prevEmmyft: /* Wednesday */2012-08-19T16:22:58Z<p><span class="autocomment">Wednesday</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Wednesday===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Wednesday===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We are all at that stage where our various pieces of DNA need putting into cells. To that end, we began transformations, and lots of them. We started using the electroporation device for the first time today, possibly making lots of cells with plasmids shoved through their walls. Or else we've just made a load of dead bacteria. Only a night in the incubator will tell.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We are all at that stage where our various pieces of DNA need putting into cells. To that end, we began transformations, and lots of them. We started using the electroporation device for the first time today, possibly making lots of cells with plasmids shoved through their walls. Or else we've just made a load of dead bacteria. Only a night in the incubator will tell.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Tom tried to induce light in some of his overnight cultures of E. coli with the lux plasmid, but found that they don't grow. We started to suspect that some of the colonies (which have been on agar plates in the fridge for almost a month now) has lost their plasmid. After consulting Jim H, we decided to do a patch test on agar plates containing arabinose. We should be able to see light on plates tomorrow.</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We also began thinking about what we're going to show at the meet up in a week. We've been invited down to the Google campus in London by the UEA team to discuss (and more importantly, present) our projects. Paul and Emmy therefore began creating a poster for it, and Oli and Tom began talking about how to order our presentation.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We also began thinking about what we're going to show at the meet up in a week. We've been invited down to the Google campus in London by the UEA team to discuss (and more importantly, present) our projects. Paul and Emmy therefore began creating a poster for it, and Oli and Tom began talking about how to order our presentation.</div></td></tr>
</table>Emmyfthttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_7&diff=74543&oldid=prevEmmyft: /* Thursday */2012-08-19T16:22:28Z<p><span class="autocomment">Thursday</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We got started on making more chemically competent cells, for use in conjunction with the electrocompetent cells. Hopefully if one fails, the other will work. Naturally, this involved the transfer of various clear liquids into other clear liquids, as well as the production of clear liquids. Such is the life of a bacterial consomm&eacute; chef.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We got started on making more chemically competent cells, for use in conjunction with the electrocompetent cells. Hopefully if one fails, the other will work. Naturally, this involved the transfer of various clear liquids into other clear liquids, as well as the production of clear liquids. Such is the life of a bacterial consomm&eacute; chef.</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Tom attempted the colony PCR for the third time with some changes in the PCR programme. Still no luck... After some research, we decided that it is probably a better idea to miniprep the plasmid out of the cells, since colony PCR seems somewhat unreliable. Tom has began preparing some overnight cultures of the E. coli.</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>More biobricks, as well, as Charlie both plated out and ordered some more. With luck these will be easier to use than the magnesium sensitive promoter.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>More biobricks, as well, as Charlie both plated out and ordered some more. With luck these will be easier to use than the magnesium sensitive promoter.</div></td></tr>
</table>Emmyfthttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_7&diff=74540&oldid=prevEmmyft: /* Tuesday */2012-08-19T16:21:26Z<p><span class="autocomment">Tuesday</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Having made all this, we did some Gibson for the riboswitch and fluorescent constructs, which *should* work this time. Certainly, we're pretty confident we have all the ingredients in the mix this time. We'll transform some cells with the resultant DNA cocktail as soon as we have some competent cells. Hopefully tomorrow then, though such efficient hopes have had a way of failing in the last couple of weeks.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Having made all this, we did some Gibson for the riboswitch and fluorescent constructs, which *should* work this time. Certainly, we're pretty confident we have all the ingredients in the mix this time. We'll transform some cells with the resultant DNA cocktail as soon as we have some competent cells. Hopefully tomorrow then, though such efficient hopes have had a way of failing in the last couple of weeks.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Tom struggled through his PCR again, retrying the split luciferase primers. No successes, alas..<del class="diffchange diffchange-inline">. After some research, we decided that it is probably a better idea to miniprep the plasmid out of the cells, since colony PCR seems somewhat unreliable. Tom has began preparing some overnight cultures of the E. coli</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Tom struggled through his PCR again, retrying the split luciferase primers. No successes, alas...</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Wednesday===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Wednesday===</div></td></tr>
</table>Emmyfthttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_7&diff=74535&oldid=prevEmmyft: /* Friday */2012-08-19T16:18:53Z<p><span class="autocomment">Friday</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>It also looks like our ampicillin has been degraded due to incorrect storage, so we made up a fresh batch and stored it. This may explain the observation that we have many small colonies of e.coli growing where they should not. Hope still remains, however, that the large colonies are still representative of successful transformants that can be used by us. We're culturing them up, though we don't imagine they will be usable.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>It also looks like our ampicillin has been degraded due to incorrect storage, so we made up a fresh batch and stored it. This may explain the observation that we have many small colonies of e.coli growing where they should not. Hope still remains, however, that the large colonies are still representative of successful transformants that can be used by us. We're culturing them up, though we don't imagine they will be usable.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Paul made up some chemically competent cells as well, which can be used in conjunction with our electrically transformable cells. We made two batches (due to miscalculations in the number of autoclaved eppendorf tubes we would need), one of them ended up going through an extra freeze-thaw cycle. <del class="diffchange diffchange-inline">We will test both of them </del>over the weekend <del class="diffchange diffchange-inline">for competency</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Paul made up some chemically competent cells as well, which can be used in conjunction with our electrically transformable cells. We made two batches (due to miscalculations in the number of autoclaved eppendorf tubes we would need), one of them ended up going through an extra freeze-thaw cycle. <ins class="diffchange diffchange-inline">He has transformed the first batch with pUC19, so if all works out we should be able to see growth on agar plates tomorrow. Then we can do further tests </ins>over the weekend.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Tom amplified up the sfGFP-ampR and pSB4K5 provided by the Haseloff Lab for Gibson control. PCR worked out very nicely and the required DNA are extracted. He will Gibson some of them together so they could be used to test our chemically competent cells.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Tom amplified up the sfGFP-ampR and pSB4K5 provided by the Haseloff Lab for Gibson control. PCR worked out very nicely and the required DNA are extracted. He will Gibson some of them together so they could be used to test our chemically competent cells.</div></td></tr>
</table>Emmyfthttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_7&diff=74532&oldid=prevEmmyft: /* Friday */2012-08-19T16:17:53Z<p><span class="autocomment">Friday</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Paul made up some chemically competent cells as well, which can be used in conjunction with our electrically transformable cells. We made two batches (due to miscalculations in the number of autoclaved eppendorf tubes we would need), one of them ended up going through an extra freeze-thaw cycle. We will test both of them over the weekend for competency.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Paul made up some chemically competent cells as well, which can be used in conjunction with our electrically transformable cells. We made two batches (due to miscalculations in the number of autoclaved eppendorf tubes we would need), one of them ended up going through an extra freeze-thaw cycle. We will test both of them over the weekend for competency.</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Tom amplified up the sfGFP-ampR and pSB4K5 provided by the Haseloff Lab for Gibson control. PCR worked out very nicely and the required DNA are extracted. He will Gibson some of them together so they could be used to test our chemically competent cells.</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Additionally, we managed to get some of the actual charactarisation started, courtesy of the plasmid sent to us by Yale. Jolyon tested the sensitivity of the riboswitch by testing the rate of lac Z transcription with a &beta;-galactosidase assay.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Additionally, we managed to get some of the actual charactarisation started, courtesy of the plasmid sent to us by Yale. Jolyon tested the sensitivity of the riboswitch by testing the rate of lac Z transcription with a &beta;-galactosidase assay.</div></td></tr>
</table>Emmyft