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Team:EPF Lausanne/Notebook
From 2010.igem.org
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Revision as of 17:13, 13 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
|
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...
|
WikiWe did a lot of work on the wiki but we haven't downloaded it yet.
|
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
|
Biobrick
|
05-08-2010
Other
|
Biobrick
|
Asaia
|
06-08-2010
Biobrick
|
Asaia
|
06-08-2010
Biobrick
|
Asaia
|
09-08-2010
Biobrick |
Asaia
|
10-08-2010
ImmunotoxineWe'll made some GLY stock of E-coli containing the immunotoxine plasmid (shiva). So today we transform E-coli with the plasmid and we plated on Amp overnight. |
BiobrickWe try to begin to construc two new biobrick today but we made mistake. We'll start again tomorrow. |
11-08-2010
ImmunotoxineWe've pick up some colony of transformed cells from yesterday and make liquid culutre of them. Tomorrow we'll make some GLY stock and a gel to control. With this stock we can begin to work safely with the immunotoxine. |
AsaiaTo test our asaia origin sequence, we take a BV with (1) asaia origin (2) Amp resistance (3) death casette and we will throw away the death casette and just transform asaia with this simpel plasmid. Today we just make a liquid culture from a GLY stock with E-coli containing this plasmid. |
BiobricksWe begin to construc a new Biobrick containing (1) Base Vector (2) Strong or Weak promotor (3) RBS. Today we made digestion, ligation and E-coli transformation. Plated overnight on Amp. |
DrosophilePersistence of bacteria in drosophilia's gut. We infected (fed) flies with Asaia. We also infected flies with persistant or pathogenic bacteria (control experiment). Then we crushed, diluted and plated infected flies after 3h hours from infection. We will plate flies after 24 and 48 hours as well and see how many colonies appear in function of time. |
12-08-2010
DrosophilesText |
BiobrickText |
02-08-2010
DrosophilesIt rain a lot today |
AsaiaDigestion/Ligation of "ORI + Resistance (Amp/Kan)" into Base Vectors |
BioBricks |
