• Design and ordering of the sequences needed for the first constructions.</p>
• Design and ordering of the sequences needed for the first constructions.</p>
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• Some technical problem have required the addition of intermediary steps that are kept silent in this summary (gel purification,…).</p>
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<h2> IV. 22/07-27/07 </h2>
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• The week begins with the negative results of the screening of the phoA and prolin::phoA bacteria. Furthermore, the control group (electropored with linearized plasmids) has numerous colonies, meaning that we may have forgotten to inhibit the circularization of the plasmid by incubating with the alkaline phosphatase.</p>
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o We thus begin the digestion of the plasmid and the inserts (phoA and phoA::proline) again. We proceed to the electroporation, goth overnight and screening of the obtained clones, but the results are all negative, once again. (25/07) After investigation, we discover that we used the wrong antibiotic (Ampicilin instead of Chloramphenicol) and that we forgot to add glucose to the medium in order to inhibit phoA overexpression and promote the bacterial growth.</p>
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• Construction of RFP :: phoA, RFP :: 2A :: phoA and RFP :: ccdB by PCR amplification.</p>
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We'll present the results week by week, in order to report the progressive advancement of our project as faithfully as possible. When possible, we'll use the structure of the $\small WetLab$ $\small\&$ $\small Methods$ section in order to clarify the link between each result and the part of the project it applies.
I. 30/06 – 06/07
• All the preliminary steps (brainstorming, organisation of the project,etc.) have already been done officiously during the year with the help of our supervisors.
• 1$^{st}$ meeting in order to organize the search of the sponsors, the creation of the wiki, and the completion of the safety form.
• A sponsoring folder has been written in French by the sponsoring team, and a first list of biotech companies and public services susceptible of offering us sponsorship has been made.
II. 07/07 – 13/07
• 2$^{nd}$ meeting to make a briefing of the coming manipulations and to find some ideas of Human Practice. Research of sponsorship is intensified.
• Familiarization with the lab and the first basic manipulations (preparation of media, dyalisis, electroporation) as well as presentation of the safe lab practices.
• Extraction of the biobricks we will use in our manipulation for cloning.
• The electrocompetent bacteria used for the cloning are MC1061 $\small E.Coli$.
• 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.
• Conservation of the transformed bacteria at -80°C (in glycerol).
• Miniprep of the RFP and GFP biobricks, midiprep of the vector AraC-pbad33 (which we will use to make our constructions).
III. 14/07-20/07
• 3rd meeting in order to report our respective advances in our quest of sponsorship, to discuss the popularization event of the Brussels Game festival, to elect delegates for the Website and to select the tracks in which we compete.
• Linearization and PCR amplification for the PSB1(A/C/K/T)3 plasmids.
• Preparation of the ccdB and Kid biobricks in the Standard 10 by PCR amplification. Cloning into bacteria and screening. We will discover next week that the cloning has failed.
• PCR amplification of phoA and prolin::phoA (prolin added via primer) from MG1655 E.coli, cloning into bacteria and screening. We will discover next week that the cloning has failed.
• Design and ordering of the sequences needed for the first constructions.
• Some technical problem have required the addition of intermediary steps that are kept silent in this summary (gel purification,…).
IV. 22/07-27/07
• The week begins with the negative results of the screening of the phoA and prolin::phoA bacteria. Furthermore, the control group (electropored with linearized plasmids) has numerous colonies, meaning that we may have forgotten to inhibit the circularization of the plasmid by incubating with the alkaline phosphatase.
o We thus begin the digestion of the plasmid and the inserts (phoA and phoA::proline) again. We proceed to the electroporation, goth overnight and screening of the obtained clones, but the results are all negative, once again. (25/07) After investigation, we discover that we used the wrong antibiotic (Ampicilin instead of Chloramphenicol) and that we forgot to add glucose to the medium in order to inhibit phoA overexpression and promote the bacterial growth.
• Construction of RFP :: phoA, RFP :: 2A :: phoA and RFP :: ccdB by PCR amplification.
"
- Université Libre de Bruxelles -
