Team:ULB-Brussels/Project/Notebook
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• Spectrophotometric verification of the well-functioning of the RFP and GFP. The chosen biobrick of GFP had no promoter, we have to fuse it with a promoter.</p> | • Spectrophotometric verification of the well-functioning of the RFP and GFP. The chosen biobrick of GFP had no promoter, we have to fuse it with a promoter.</p> | ||
• Conservation of the transformed bacteria at -80°C (in glycerol).</p> | • Conservation of the transformed bacteria at -80°C (in glycerol).</p> | ||
- | • Miniprep of the RFP and GFP biobricks, midiprep of the vector AraC- | + | • Miniprep of the RFP and GFP biobricks, midiprep of the vector AraC-pBAD33 (which we will use to make our constructions).</p> |
<h2> III. 14/07-20/07 </h2> | <h2> III. 14/07-20/07 </h2> | ||
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• We restarted the PCR amplification for the constructions in $\SCere$ (RFP :: p2A :: GFP, RFP :: p2A :: GFP, Kid) and the amplification of the RFP(RFP2) gene for the construction RFP :: p2A :: phoA in $\EColi$. This time, the result was positive (29/07/14).</p> | • We restarted the PCR amplification for the constructions in $\SCere$ (RFP :: p2A :: GFP, RFP :: p2A :: GFP, Kid) and the amplification of the RFP(RFP2) gene for the construction RFP :: p2A :: phoA in $\EColi$. This time, the result was positive (29/07/14).</p> | ||
• We lost the RFP and ccdB segments for the construction RFP::ccdB, or forgot that we had amplified them. We amplified them a 2nd time (30/07/14)</p> | • We lost the RFP and ccdB segments for the construction RFP::ccdB, or forgot that we had amplified them. We amplified them a 2nd time (30/07/14)</p> | ||
- | • We digested | + | • We digested pBAD33 with the restriction enzyme sal1 in order to begin our constructions using the In-Fusion kit. The concentration was weak but the digestion seems to have functioned. We also digested pGAl1 with Hind3 for the same reason (31/07/14). |
- | • Purification of the RFP:ccdB and ccdB- | + | • Purification of the RFP:ccdB and ccdB-pBAD segment for their fusion in RFP-ccdB-pBAD33 using the kit In-Fusion.</p> |
- | • Making of the | + | • Making of the pBAD33 ::RFP ::phoA and pBAD33 ::RFP ::ccdB constructions using the In-Fusion kit. Electroporation and overnight culture (01/08/14). We later screened the colonies to observe that the manipulation had failed (04/08/14).</p> |
<!-- Add Figure 7 to 9 --> | <!-- Add Figure 7 to 9 --> | ||
• In the same time, we continuated the TA system modelling, extended in a case of the production of a PI in general and we drew a first diagram illustrating the model.</p> | • In the same time, we continuated the TA system modelling, extended in a case of the production of a PI in general and we drew a first diagram illustrating the model.</p> | ||
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• Due to miscommunication and bad labelling, we had to remake most of the segment that had been previously made but somehow lost. </p> | • Due to miscommunication and bad labelling, we had to remake most of the segment that had been previously made but somehow lost. </p> | ||
• End of the TA system modelling, because another model is suggested and the dynamics of the CcdBA complexation seemed not be theoretically understood in the literature. Begin to search methods to construct a model for the 2A peptid effect.</p> | • End of the TA system modelling, because another model is suggested and the dynamics of the CcdBA complexation seemed not be theoretically understood in the literature. Begin to search methods to construct a model for the 2A peptid effect.</p> | ||
- | • The whole week in Lab has been spent carefully digesting | + | • The whole week in Lab has been spent carefully digesting pBAD33 and pGAL1, regrowing proline::phoA colonies, re-amplificating the segment we had lost or depleted and purifying them (either on gel or on column). This took time since several of these steps have failed, so we had to restart so often that we were not more advanced at the end of the week.</p> |
o For example, when the digestion worked properly, something went wrong during the purification and everything was lost. We changed our protocols in small ways several time by listening to the advices of the PhD students in the lab.</p> | o For example, when the digestion worked properly, something went wrong during the purification and everything was lost. We changed our protocols in small ways several time by listening to the advices of the PhD students in the lab.</p> | ||
o At the end of the week, we had sent 2 phoA plasmids and 1 proline::phoA plasmid to the sequencing, but not much more.</p> | o At the end of the week, we had sent 2 phoA plasmids and 1 proline::phoA plasmid to the sequencing, but not much more.</p> | ||
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<h2> VII. 11/08/14- 17/08/14 </h2> | <h2> VII. 11/08/14- 17/08/14 </h2> | ||
• We continued this week what we had begun the week before: all our time has been consumed by digestions that did not want to work anymore (although we never had digestion problem in July) and purification that removed the DNA as well as the impurities.</p> | • We continued this week what we had begun the week before: all our time has been consumed by digestions that did not want to work anymore (although we never had digestion problem in July) and purification that removed the DNA as well as the impurities.</p> | ||
- | o At the end of the week, we had launched a In-Fusion construction of RFP::p2A::GFP in pGAL, and RFP::phoA and RFP::ccdB were purified and available for the In-Fusion kit but the | + | o At the end of the week, we had launched a In-Fusion construction of RFP::p2A::GFP in pGAL, and RFP::phoA and RFP::ccdB were purified and available for the In-Fusion kit but the pBAD33 plasmids were not ready.</p> |
- | o We tested our restriction enzyme, but apparently they were functional, so we decided that the | + | o We tested our restriction enzyme, but apparently they were functional, so we decided that the pBAD33 plasmid we were using were somehow damaged.</p> |
• We had our Human Practice event at the Brussels Game Festival, which was a success.</p> | • We had our Human Practice event at the Brussels Game Festival, which was a success.</p> | ||
<!-- Add Figure 12 --> | <!-- Add Figure 12 --> | ||
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<h2> VIII. 18/08/14-24/08/14 </h2> | <h2> VIII. 18/08/14-24/08/14 </h2> | ||
The screening of the colonies of bacteria electropored by the In-Fusion product was not satisfying. It is our second negative result with the In-Fusion kit, thus we transform by lithium acetate and try to exploit the homolog recombining property (in vivo, this is equivalent as with In-Fusion kit) of the yeast to make our constructions. See protocol: “Yeast transformation by lithium acetate». Our results were not satisfactory.</p> | The screening of the colonies of bacteria electropored by the In-Fusion product was not satisfying. It is our second negative result with the In-Fusion kit, thus we transform by lithium acetate and try to exploit the homolog recombining property (in vivo, this is equivalent as with In-Fusion kit) of the yeast to make our constructions. See protocol: “Yeast transformation by lithium acetate». Our results were not satisfactory.</p> | ||
- | The | + | The pBAD33::phoA & pBAD33::phoA::proline sequences are now available.</p> |
In chromogenic zn, test fn ok $\rightarrow$ phoA works, but one really better than the other; we try to verify if phoA::proline doesn't works (mutation vs proline).</p> | In chromogenic zn, test fn ok $\rightarrow$ phoA works, but one really better than the other; we try to verify if phoA::proline doesn't works (mutation vs proline).</p> | ||
<h2> IX. 25/08/14-31/08/14 </h2> | <h2> IX. 25/08/14-31/08/14 </h2> | ||
- | New | + | New pBAD33::phoA::proline construction $\rightarrow$ 4 colonies were survived.</p> |
Extra functional test: the presence of proline deosn't stop the activity oh phoA.</p> | Extra functional test: the presence of proline deosn't stop the activity oh phoA.</p> | ||
- | We also discussed the problem of purification of plasmid | + | We also discussed the problem of purification of plasmid pBAD33 digested. We hypothesized that the digestion by the enzyme Sal1 results in a secondary structure of the plasmid such that it remains localized at the column. To prove this, we determined that a sample of digested pBAD33 was well concentrated in plasmid. We then performed the normal purification, except that at each step rather than discard the supernatant we have preserved to ascertain the presence of the plasmid. We also conducted several elutions. We have found the presence of the plasmid in any of these fractions and only at low concentration in elution fractions. This result supports our hypothesis and allows to explain the difficulties we have met.</p> |
<h3> August summary </h3> | <h3> August summary </h3> | ||
</p>During this month, despite a best skill, we encountered many difficulties that have hampered the progress of our project. Simple manipulations, yet well mastered, proved unsuccessful to numerous occasions.</p> | </p>During this month, despite a best skill, we encountered many difficulties that have hampered the progress of our project. Simple manipulations, yet well mastered, proved unsuccessful to numerous occasions.</p> | ||
</p>Although the situation was very frustrating, it has nevertheless allowed us to deal with the vagaries of the research profession and to learn to think where problems come from and also how resolve them.</p> | </p>Although the situation was very frustrating, it has nevertheless allowed us to deal with the vagaries of the research profession and to learn to think where problems come from and also how resolve them.</p> | ||
- | </p>We also had the pleasure to get positive results for our constructions, in this case the BioBrick ccdb and the control test | + | </p>We also had the pleasure to get positive results for our constructions, in this case the BioBrick ccdb and the control test pBAD::phoA::proline. However, these results have rubbed shoulders the arrival of September inducting the halting of the lab manipulations.</p> |
</section> | </section> |
Revision as of 22:05, 10 October 2014
$~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ \newcommand{\MyColi}{{\small Mighty\hspace{0.12cm}Coli}} \newcommand{\Stabi}{\small Stabi}$ $\newcommand{\EColi}{\small E.coli} \newcommand{\SCere}{\small S.cerevisae}\\[0cm] ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ \newcommand{\PI}{\small PI}$ $\newcommand{\Igo}{\Large\mathcal{I}} \newcommand{\Tgo}{\Large\mathcal{T}} \newcommand{\Ogo}{\Large\mathcal{O}} ~$
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