This is our final, final week of lab work. It is also the week where we finished characterising BM and MB. In this week we proved that it did what we created it to do: increase flexibility by working in both mammalian and bacterial cells. We created BM and MB as we were worried orientations would affect the function but through this weeks experiments we now know that the orientation does affect it but only it's efficiency. We also characterised them quantitatively using flourometry, flow cytometry and FACs. It's been an awesome awesome week. The last 11 weeks have been so worth it. The saddest moment was leaving the labs for the final time.
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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, flow cytometry and FACs. 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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[[File:NRPFluorescence.jpeg | 300 px | thumbnail | Figure: The peletted cells of BM attached to RFP and CFP fluorescing after growth in potassium nitrate.]]
[[File:NRPFluorescence.jpeg | 300 px | thumbnail | Figure: The peletted cells of BM attached to RFP and CFP fluorescing after growth in potassium nitrate.]]
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We Miniprepped MB attached to CFP and many BM's attached to RFP and CFP that had been grown from different plates. The concentrations were very low. These will either be discarded if readings are low after renanodropping as there may be a slight issue with the nano drop machine used.
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We miniprepped MB attached to CFP and many versions of BM attached to RFP and CFP, that had been grown from different plates. The DNA concentrations were very low so the samples will either be discarded if readings are still low after renanodropping, as there may be a slight issue with the nano drop machine used.
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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 have successful ligations and BioBricks. However, as the miniprepping was unsuccessful, we re-innoculated and will try again tomorrow.
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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.
==Day 2 (18/09/12)==
==Day 2 (18/09/12)==
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We carried out flow cytometry of BM with RFP and then FACS of BM-RFP and MB-CFP transformed ''E.coli''. The full results are on the experiments page. We also carried out a pilot study for the flouorometer study on BM-RFP and MB-CFP. We used only a few concentrations of potassium nitrate: 0mM, 1mM and 10mM. The results were extremely promising. The wavescans were as we expected them to be. The 0mM wavescan showed the lowest OD readings and the highest were the 10mM. There was no overlap between any of the wavescans.
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We carried out flow cytometry of BM with RFP and following up with FAC's of BM-RFP and MB-CFP transformed ''E.coli''. The full results are on the [https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments experiments page] and protocols are written up [https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol here]. We also preformed a pilot for the flouorometer study on BM-RFP and MB-CFP. We used only a selected few concentrations of potassium nitrate: 0mM, 1mM and 10mM. The results were extremely promising [https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments experiments page]. The wavescans showed the results as we expected them to be. The data at 0mM showed the lowest OD readings and the highest were found at 10mM. There was no overlap between any of the wavescans.
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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 experiments page.
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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].
==Day 3 (19/09/12)==
==Day 3 (19/09/12)==
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We identified the Nir A promoter as a promoter that will specifically sense the concentration of nitrates.
We identified the Nir A promoter as a promoter that will specifically sense the concentration of nitrates.
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With the promising results from the piot study on CFP and RFP's using the fluorometer, we did the full experiment using MB-CFP with multiple concentrations of potassium nitrate: 0mM, 5mM, 15mM and 20mM. For full details check the experiments.
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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].
==Day 4 (20/09/12)==
==Day 4 (20/09/12)==
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Today we sent off the rest of our BioBricks to iGEM! These were:
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Today we sent off our latest validated, sequenced and proven BioBricks to iGEM! These were:
BBa_K774002 (Comparator Circuit Construct 1)
BBa_K774002 (Comparator Circuit Construct 1)
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We also set up the experiment for tomorrow's fluorometer study. We inoculated 5 tubes of media with B-M + RFP and 5 tubes of media with M-B + RFP, we then added potassium nitrate to make them up to 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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We also set up the preparations for tomorrow's fluorometer study. We inoculated 5 tubes of media with B-M + RFP and 5 tubes of media with M-B + 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.
==Day 5 (21/09/12)==
==Day 5 (21/09/12)==
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With promising results from the piot study on CFP and RFP's and the MB-CFP study on Wednesday, we continued this and we did the full experiment using BM-RFP with multiple concentrations.
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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].
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, flow cytometry and FACs. 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.
Figure: The peletted cells of BM attached to RFP and CFP fluorescing after growth in potassium nitrate.
We miniprepped MB attached to CFP and many versions of BM attached to RFP and CFP, that had been grown from different plates. The DNA concentrations were very low so the samples will either be discarded if readings are still low after renanodropping, as there may be a slight issue with the nano drop machine used.
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.
Day 2 (18/09/12)
We carried out flow cytometry of BM with RFP and following up with FAC's of BM-RFP and MB-CFP transformed E.coli. The full results are on the experiments page and protocols are written up here. We also preformed a pilot for the flouorometer study on BM-RFP and MB-CFP. We used only a selected few concentrations of potassium nitrate: 0mM, 1mM and 10mM. The results were extremely promising experiments page. The wavescans showed the results as we expected them to be. The data at 0mM showed the lowest OD readings and the highest were found at 10mM. There was no overlap between any of the wavescans.
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 experiments page and the protocol can be found here.
Day 3 (19/09/12)
We identified the Nir A promoter as a promoter that will specifically sense the concentration of nitrates.
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 experiments page.
Day 4 (20/09/12)
Today we sent off our latest validated, sequenced and proven BioBricks to iGEM! These were:
BBa_K774002 (Comparator Circuit Construct 1)
BBa_K774003 (Comparator Circuit Construct 2)
BBa_K774004 (B-M + eCFP)
BBa_K774005 (B-M + RFP)
BBa_K774006 (M-B + eCFP)
BBa_K774007 (M-B + RFP)
We also set up the preparations for tomorrow's fluorometer study. We inoculated 5 tubes of media with B-M + RFP and 5 tubes of media with M-B + 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.
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 experiments page.
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