Week 1

12-07-2010

• OD measurement of Asaia’s culture
• Chemical competence for E.Coli DB3.1 (step : Day 3)
• PCR (Asaia ORI + primers)=12-07-2010=
• 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

13-07-2010

• 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

14-07-2010

• Run a gel for the PCR of the previous day -> worked
• Purification of PCR’s product -> ok
• Preparation of the BBa_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
• Culture of Asaia + Kan

15-07-2010

• 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
• Electrophoresis of resistance fragments (Works!!!) and extraction
• Purification of extracted DNA from agarose gel

16-07-2010

• 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 (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