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$~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ \newcommand{\MyColi}{{\small Mighty\hspace{0.12cm}Coli}} \newcommand{\Stabi}{\small Stabi}$ $\newcommand{\EColi}{\small E.coli} \newcommand{\SCere}{\small S.cerevisae}\\[0cm] ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ \newcommand{\PI}{\small PI}$ $\newcommand{\Igo}{\Large\mathcal{I}} \newcommand{\Tgo}{\Large\mathcal{T}} \newcommand{\Ogo}{\Large\mathcal{O}} ~$

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Previous Years 2012 : InteGreator 2011 : Pindel 2010 : HydroColi 2009 : GluColi

- Université Libre de Bruxelles - Results Retrieved from "http://2014.igem.org/Team:ULB-Brussels/Project/Results" Recent changes What links here Related changes Special pages My preferences Printable version Permanent link Privacy policy Disclaimers New Results 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 :: p2A :: phoA and RFP :: ccdB by PCR amplification. o The templates for phoA, RFP and ccdB have respectively been found in MG1065 E.coli, MC1061 E.coli infected by a RFP biobrick from the iGEM, and in a sample coming directly from IDT DNA technologies. o We failed the experiment completely, restarted, and this time we managed to amplify and successfully purify all the segments except phoA2 and RFP2. • Screening of the Kid and ccdB clones for the making of biobricks. (23/07) KID has not been integrated into the colonies; on the other hand, was successfully cloned in several bacteria. o We restart the amplification of KID from the beginning, and make an overnight culture of the colonies containing ccdB. We then store these cultures at -80°C in glycerol and, in parallel; we make a mini-prep out of it in order to send it to sequencing. o The second try of the amplification of KID is also negative (25/07/14), even after considering more colonies (28/07/14). • The synthetic sequences ordered by our supervisors for the constructions in S. cerevisae have arrived. We thus proceed to the PCR amplification to build our constructions. The manipulation has completely failed (24/07/14), we will have to start again. V. 28/07-03/08 • After having observed the failure of the screening of phoA, proline::phoA and Kid, we decided to abandon the fabrication of the Kid biobrick to focus on $\MyCOli$ (results not shown). We restarted the phoA and prolin::phoA manipulation several times from intermediary steps, and we eventually restarted it one last time from the very beginning, by amplifying the gene phoA from MG1655 E.coli (28/07/14). o We later discovered that we did not use the proper annealing temperature during the screening, nor did we adjust the duration of the elongation phase of the PCR to the length of phoA. It means that we might have grown phoA and phoA+P bacteria without noticing it. o This time we managed to find 1 colony possessing phoA but still none with prolin::phoA (05/08). • We restarted the PCR amplification for the constructions in S.cerevisiae (RFP :: p2A :: GFP, RFP :: p2A :: GFP, Kid) and the amplification of the RFP(RFP2) gene for the construction RFP :: p2A :: phoA in E.coli. This time, the result was positive (29/07/14). • 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) • 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-pbad segment for their fusion in RFP-CCDB-pBAD33 using the kit In-Fusion (31/07/14). • 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). < WetLab & Methods