Team:NRP-UEA-Norwich/Week11
From 2012.igem.org
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- | This was our final, final week of lab work. It is also the week in which we concluded the characterisation of BM and MB. We proved that it did indeed fulfil the expectations we had and that it functioned in the way we designed it to. We created BM/MB in both orientations as we were worried that it would affect the function. Through this weeks experiments, it was proven that the orientation does have an affect but only on the efficiency of the promoter. We also characterised them quantitatively using flourometry | + | This was our final, final week of lab work. It is also the week in which we concluded the characterisation of BM and MB. We proved that it did indeed fulfil the expectations we had and that it functioned in the way we designed it to. We created BM/MB in both orientations as we were worried that it would affect the function. Through this weeks experiments, it was proven that the orientation does have an affect but only on the efficiency of the promoter. We also characterised them quantitatively using flourometry and flow cytometry. It was an awesome, awesome week with everything coming together perfectly. The last 11 weeks have been leading up to the results we obtained and it was worth it. But all good things come to end and the saddest moment was leaving lab for the final time. |
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The cell cultures of the BM ligated to CFP and RFP were pelletted and viewed under UV light to check for fluorescence. We found that when grown in the presence of potassium nitrate, they did as expect glow and hence we suspect the ligation to have been successful and that we have created new, functional BioBricks. However, as the miniprepping was unsuccessful, we re-innoculated and plan to try again tomorrow. | The cell cultures of the BM ligated to CFP and RFP were pelletted and viewed under UV light to check for fluorescence. We found that when grown in the presence of potassium nitrate, they did as expect glow and hence we suspect the ligation to have been successful and that we have created new, functional BioBricks. However, as the miniprepping was unsuccessful, we re-innoculated and plan to try again tomorrow. | ||
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==Day 2 (18/09/12)== | ==Day 2 (18/09/12)== | ||
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To continue the MB-CFP transfection into MCF7 (Human breast cancer cells), the cells were treated with SNAP (an Nitric Oxide donor) and imaged using a Zeiss CCD2 inverted microscope to detect CFP expression. All the images and details will be on the [https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments experiments page] and the protocol can be found [https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol here]. | To continue the MB-CFP transfection into MCF7 (Human breast cancer cells), the cells were treated with SNAP (an Nitric Oxide donor) and imaged using a Zeiss CCD2 inverted microscope to detect CFP expression. All the images and details will be on the [https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments experiments page] and the protocol can be found [https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol here]. | ||
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==Day 3 (19/09/12)== | ==Day 3 (19/09/12)== | ||
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With the promising results from the piot study on CFP and RFP's using the fluorometer yesterday, we conducted the full experiment using MB-CFP with multiple concentrations of potassium nitrate: 0mM, 5mM, 15mM and 20mM. For full details check the [https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments experiments page]. | With the promising results from the piot study on CFP and RFP's using the fluorometer yesterday, we conducted the full experiment using MB-CFP with multiple concentrations of potassium nitrate: 0mM, 5mM, 15mM and 20mM. For full details check the [https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments experiments page]. | ||
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==Day 4 (20/09/12)== | ==Day 4 (20/09/12)== | ||
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BBa_K774003 (Comparator Circuit Construct 2) | BBa_K774003 (Comparator Circuit Construct 2) | ||
- | BBa_K774004 ( | + | BBa_K774004 (BM + eCFP) |
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+ | BBa_K774005 (BM + RFP) | ||
- | + | BBa_K774006 (MB + eCFP) | |
- | + | BBa_K774007 (MB + RFP) | |
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+ | We also set up the preparations for tomorrow's fluorometer study. We inoculated 5 tubes of media with BM + RFP and 5 tubes of media with MB + RFP. Then potassium nitrate was added to make them up concentrations of 0 mM, 5 mM, 10 mM, 15 mM and 20 mM. These were grown overnight in the 37 ºC shaker. | ||
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==Day 5 (21/09/12)== | ==Day 5 (21/09/12)== | ||
With promising results from the piot study on CFP and RFP's and the MB-CFP study on Wednesday, we further pursued this route and conducted full experiment using BM-RFP with varying potassium nitrate concentrations. The final results of our iGEM 2012 lab work can be found on the [https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments experiments page]. | With promising results from the piot study on CFP and RFP's and the MB-CFP study on Wednesday, we further pursued this route and conducted full experiment using BM-RFP with varying potassium nitrate concentrations. The final results of our iGEM 2012 lab work can be found on the [https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments experiments page]. |
Latest revision as of 02:06, 27 September 2012