How to use the Notebook

template of a day

Calendar

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

19-07-2010

• Results of the transformation checked: successful!!
• Amplification of colonies (goal to do a miniprep of the plasmids --ori-- and --ori-resistance--)
• Results of plating of asaia culture (where we hope to recover the WT) checked: Asaia cultures grew on Kan plates also! Checked colonies for fluorescance under the microscope: no fluorescance --> Very unexpected! Don't know reason yet.
• Re-grow the colonies. Multiple cycles: hope that we get WT.
• Preparation of Asaia cultures for Lemaitre experiments/WT/Competence...
• Preparation of SOC medium and GLY medium
• Autoclaving of media and flasks

20-07-2010

• Plating Asaia to get WT (once again)
• Making HEPES Buffer for the Competence of Asaia
• Diluted the culture to get a 25ml culture(1:20) according to Asaia competence protocol
• OD curve plotted, Doubling time calculated
• No single colonies from the re-growing trial --> replating properly on separate plates
• Preparation of LB medium mixed with Kan/Amp/Cm and GLY medium mixed with <kan
• Miniprep of Origin + Resistance
• Miniprep of Origin alone.
• Miniprep of base vector BBa_151020
• Concentration measurments of miniprep products
• Glycerol stock of BB plasmid and Base Vector made

21-07-2010

• Measure OD of overnight culture of Asaia for competence --> value was too high
• Diluted culture to 0.3 OD, left in 30 degree incubator for 2 hours, OD of 0.6 measured --> start with competence
• Asaia competence
• Digestion of BB's produced and of the Base vector
• Agar plates with Kan/Tet/Cm/Amp made
• Prepared Gel with the products of the digestion to check if the fragments are as expected
• Prepare Tet stock (from powder)

22-07-2010

• Gel with products of digestion run --> only one of the base vector samples and a fragment with the origin and a Kan resistance worked. The others probably did not wok since the concentration of DNA was too low.
• Colonies picked and suspension cultures started. For making a miniprep tomorrow morning.
• Gel cut, Gel purification made
• New idea: PCR run on the product of the miniprep.
• Ligation of Base Vector with Origin+Kan
• Asaia preculture for OD growth (different pH) measurements
• Gly medium with pH 2,3,4,5,6,7.
• Research
• Replating of Asaia (Replating III) to get the WT (one day!!)

23-07-2010

• OD measurement growth curve in function of pH
• new DNA miniprep to extract Ori+AB_Res (the last one failed...)
• Lyse-n-Go protocol to do the ligase (and to check the insertion)
• transformation of the only digestion from yesterday that didn't failed (Kan)
• meeting with Onya for mosquito experiments
• preparation of 4L of Gly for LeMaitre experiments

26-07-2010

• Lemaître experiment: 400 ml cultures of GFP Asaia, Ecc15 and P.Entomophila
• Lemaître experiment: separating male and female drosophila
• Transformation of the Base Vector with -Kan-ori- insert into E.Coli DB3.1
• PCR of LovTab as a positive control to check if the PCR works (with own and Henrike's dNTP's) --> Run gel
• Test-digestion of some of the Mini-preps (Ori alone, Ori-Amp) --> Run gel
• Recovered the wildtype Asaia worked!!
• WT Asaia strains arrived. Culture started

27-07-2010

• Ligation C3/A3 + Ori + resistance (Kan, Amp, Tet, Cm)
• transformation of the ligation products into E.Coli DH5a
• Plating of transformed cells with selection of the vector and the insert
• Colony PCR of E.Coli DB3.1 containing BaseVector + Ori + Kan --> running gel (twice, the first time failed)
• Preculture of (maybe!!) WT Asaia for competence protocol
• Sorting of male/female flies
• Culture of 400 ml of different species of bacteria.

28-07-2010

• Analysis of plating of transformed cells
• Gel for check the Colony PCR
• 250 ml Gly medium
• OD measurement of preculture for competent Asaia --> too high. Start a new one!!!
• Competent Asaia and stocking @-80°C
• Plating of transformed E.Coli for double check
• preparation of the pellets for infection with 3 different species of bacteria + OD measurements
• preparation of tubes with 15 female flies
• begin of the survival experiment: infection of flies --> check of the flies after 2h

29-07-2010

• Results from plating of transformed E.Coli for double check and miniprep from some of them
• Digestion with different restriction enzymes to check if we get the right length
• Counting of dead flies one day after the infection
• Observation of fluorescence of infected flies under the microscope

30-07-2010

• Running a gel of the digested products of miniprep (29.07) --> bad results
• Miniprep for the last 17 samples of replating
• Digestion of 5 samples with 3 restriction enzymes to check if we get the fragments we expect
• Running a gel for the digestion products
• Counting of dead flies two days after the infection
• Observation of fluorescence of infected flies under the microscope

02-08-2010