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Team:EPF Lausanne/Notebook
From 2010.igem.org
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==Immunotoxin== | ==Immunotoxin== | ||
Henrike verified the primer sequences for us and ordered them --> they should arrive on Friday or Monday. | Henrike verified the primer sequences for us and ordered them --> they should arrive on Friday or Monday. | ||
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== Asaia == | == Asaia == | ||
* The asaia transformation we let in the incubator since 02-08 are now showing colonies! As expected and verified under the fluorescent microscope, they express GFP. | * The asaia transformation we let in the incubator since 02-08 are now showing colonies! As expected and verified under the fluorescent microscope, they express GFP. | ||
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* Transformed Asaia with the pB129421248??? plasmid from E. coli to see whether it works in Asaia. | * Transformed Asaia with the pB129421248??? plasmid from E. coli to see whether it works in Asaia. | ||
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== Other == | == Other == | ||
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== Biobrick == | == Biobrick == | ||
Revision as of 19:27, 6 August 2010
How to use the Notebook
This is a NOTEbook and not a LABbook. Here you can describe what you have done during your exciting day. If you want to have more details you can look in the IRL Labbook. Use ctrl+F to find something ;)
template of the day
For each new day in the lab you must create a new date in the notebook. To do so, copy-paste the following template:
= 02-08-2010 =
{| cellspacing="15"
| style="width: 33.33%" valign="top" |
== Other ==
It rain a lot today
| style="width: 33.33%" valign="top" |
== Asaia ==
Today I speak with an asaia and she answer to me ^^
| style="width: 33.33%" valign="top" |
== Drosophile ==
|}
|
Here are some useful explanations : 1. The date format is important! 2. Just after the date, put the following two lines :
{| cellspacing="15"
| style="width: 33.33%" valign="top" |
3. Beetween each subtitle, put this line : | style="width: 33.33%" valign="top" | 4. To end the day just put : |} 5. Of course the text "width:33.33%" must be adapted on how many subtitle you have (for instance 4 subtitles = 25%) |
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
Other
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AsaiaToday we transform competent Asaia with a GFP + kanamycin (resistance) following this protocols. We plate it and we'll waiting 2 days to see if it works. we do digestion to create our first biobrick. We will make the ligation and the transformation in E-Coli tomorrow. |
Drosophilewe count the drosophile and take photos with the fluorecense microscope. In some tubes the filter has detached so the expermiment for those tube are finished. |
03-08-2010
ImmunotoxinWe have designed primers that overlap, generating a promoter and ribosome binding site. We want to put this in a vector which would be our basis to add other genes, for example our immunotoxin. We designed a strong promoter+RBS and a weak one, just in case the strong one generates too much immunotoxin and kills the bacteria...
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WikiWe did a lot of work on the wiki but we haven't downloaded it yet. Tomorrow you will see!
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AsaiaWe keep counting the drosophila and taking pictures with the GFP filter.
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Biobrick
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04-08-2010
ImmunotoxinHenrike verified the primer sequences for us and ordered them --> they should arrive on Friday or Monday. |
Asaia
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Other
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Biobrick
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05-08-2010
Other
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Biobrick
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Asaia
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06-08-2010
Biobrick
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Asaia
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