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[https://2010.igem.org/Team:EPF_Lausanne home] <h3>Monday 12.07.2010</h3> *OD measurement of Asaia’s culture *Chemical competence for E.Coli DB3.1 (step : Day 3) *PCR (Asaia ORI + primers) *Made GLY agar plates without antibiotics -> failed (medium was too old) *Asaia O/N culture without antibiotics in order to recover some WT *Text for the sponsor <h3>Tuesday 13.07.2010 </h3> *Chemical competence for E.Coli DB3.1 (step : Day 4) -> stored at -80°C *Preparation of GLY Medium / LB Medium / SOC Medium / TAE 1X / TAE 0.5X *Run a gel for the PCR -> failed (mix of two different kits) *Preparation of plates (GLY Agar / LB+Kan Agar / LB+Amp Agar) *Learned to autoclave *Restarted PCR (Asaia ORI + primers) *Plated Asaia (O/N culture) *Choice of Antibiotics Biobricks resistance (Kan / Amp / Tet / Chl) from the iGem Kit and preparation of them (streak out) *Transformation of E.coli DB3.1 with pUC19 to check its competence *Transformation of E.coli DH5 with p1003 (Kan) / p1002 (Amp) / p1004 (Chl) / p1005 (Tet) *E.coli DB3.1 and DH5 were plated on LB+Amp Agar plates (Kan was plated on LB+Kan Agar plate) *Ordered material *Wiki brainstorming *Protocols printing and organization <h3>Wednesday 14.07.2010</h3> *Run a gel for the PCR of the previous day -> worked *Purification of PCR’s product -> ok *Preparation of the BBα_151020 (streak out) *Competence Calculation of E.Coli DB3.1 (+pUC10, Amp) -> ok *Look at Asaia with microscope -> we have cells (pictures) *Growth Curve for Asaia (OD measurement each hour) -> both manually (ok) + with the plate reader *Plated Asaia to recover some WT (streak out) *Preparation of Asaia Culture (for Lemaître experiment) with Kan *Protocol LateX template *O/N culture of the E.coli DH5 transformed with the BB Antibiotics resistance (4x) -> miniprep *Learning HTML for the wiki *Text for the project presentation due to iGem *Writing Protocols of basic procedures for the wiki page *Maxi-prep of Asaia + Kan <h3>Thursday 15.07.2010</h3> *MaxiPrep of Lemaître’s culture *Purification of the BB resistances from the E.Coli cultures *Protocol for cloning the Asaia Vector *Enzyme digestion of the BB resistances and Asaia_ORI *Cloning (Asaia_ORI + Vector/ Asaia_ORI + BB + Vector) *Wiki text *Preparation of LB agar + Cm *PCR of resistance (Works!!!) and extraction *Purification of extracted DNA from agarose gel <h3>Friday 16.07.2010</h3> *ligation Asaia origin BB + vector *ligation Asaia origin BB + Kan + vector *ligation Asaia origin BB + Amp + vector *ligation Asaia origin BB + Cm + vector *ligation Asaia origin BB + Tet + vector *Extraction of the OD curve data ([https://2010.igem.org/Image:Results_OD_asaia.xls values]) *Replating Asaia on plates with and without kanamicin in order to recover the WT Asaia (only WT do not grow on kanamicin) *Feeding and infection of Drosophila (WT and immunodeficient redish mutants) with bacteria Ecc15 and Asaia in Prof. Lemaitre Lab *Fluorescence microscopy -> presence of fluorescence in the body/gut as expected *Transformation of E-Coli with the ligation products and plating the transformed bacteria

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