Team:EPF Lausanne/Notebook

From 2010.igem.org

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*11h32 Solange lives home
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Revision as of 09:32, 6 August 2010



Contents

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

Week Monday Tuesday Wednesday Thursday Friday Satursday Sunday
28 12-07-2010 13-07-2010 14-07-2010 15-07-2010 16-07-2010 17-07-2010 18-07-2010
29 19-07-2010 20-07-2010 21-07-2010 22-07-2010 23-07-2010 24-07-2010 25-07-2010
30 26-07-2010 27-07-2010 28-07-2010 29-07-2010 30-07-2010 31-07-2010 01-08-2010
31 02-08-2010 03-08-2010 04-08-2010 05-08-2010 06-08-2010 07-08-2010 08-08-2010
32 09-08-2010 10-08-2010 11-08-2010 12-08-2010 13-08-2010 14-08-2010 15-08-2010
33 16-08-2010 17-08-2010 18-08-2010 19-08-2010 20-08-2010 21-08-2010 22-08-2010
34 23-08-2010 24-08-2010 25-08-2010 26-08-2010 27-08-2010 28-08-2010 29-08-2010
35 30-08-2010 31-08-2010 01-09-2010 02-09-2010 03-09-2010 04-09-2010 05-09-2010
36 06-09-2010 07-09-2010 08-09-2010 09-09-2010 10-09-2010 11-09-2010 12-09-2010
37 13-09-2010 14-09-2010 15-09-2010 16-09-2010 17-09-2010 18-09-2010 19-09-2010
38 20-09-2010 21-09-2010 22-09-2010 23-09-2010 24-09-2010 25-09-2010 26-09-2010
39 27-09-2010 28-09-2010 29-09-2010 30-09-2010 01-10-2010 02-10-2010 03-10-2010
40 04-10-2010 05-10-2010 06-10-2010 07-10-2010 08-10-2010 09-10-2010 10-10-2010
41 11-10-2010 12-10-2010 13-10-2010 14-10-2010 15-10-2010 16-10-2010 17-10-2010
42 18-10-2010 19-10-2010 20-10-2010 21-10-2010 22-10-2010 23-10-2010 24-10-2010
43 25-10-2010 26-10-2010 27-10-2010 28-10-2010 29-10-2010 30-10-2010 31-10-2010
44 01-11-2010 02-11-2010 03-11-2010 04-11-2010 05-11-2010 06-11-2010 07-11-2010

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

We make some plate with GLY medium and Agar.

Asaia

Today 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.

Drosophile

we 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

Immunotoxin

We 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...


Wiki

We did a lot of work on the wiki but we haven't downloaded it yet. Tomorrow you will see!


Asaia

We keep counting the drosophila and taking pictures with the GFP filter.


Biobrick

  • Digestion of the plasmid containing the origin + one resitance (either Amp, Kan or Ch) and digestion of the vector i51020
  • Ligation ofthe the different parts (one insert in one back bone vector) => origin+resistance+backbone_vector_for_biobrick
  • Plating over night => we obtain something on the plate for the ori + amp nothing on the other

04-08-2010

Immunotoxin

Henrike verified the primer sequences for us and ordered them --> they should arrive on Friday or Monday.

Wiki

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.
  • Transformed Asaia with the pB129421248??? plasmid from E. coli to see whether it works in Asaia.

Other

  • Continued counting the dead Drosophilae
  • Made 2l of liquid gly medium
  • Put 2 5ml cultures of recovered wt-Asaia in the incubator to make competent cells


Biobrick

  • Make 5 ml miniprep to verify the ori + ch + i5010
  • Make negativ control for the ori + ch + i5010 on Lb+Amp plate (as the ori + amp were in the A3 backbonevector)

05-08-2010

Wiki

Other

  • Continued counting the dead Drosophilae and taking pictures.
  • Measured the OD of the two 5ml wt-Asaia cultures (4.1 and 4.7),diluted the cultures to obtain a solution with OD 0.6 tomorrow morning and finally make competent cells

Biobrick

  • The previous ligation didn't work at all, so we made a new ligation with ori + res + i5010
  • We replated the collonies that had well grown on the plate the 28.7 to keep them in glycerol

Asaia

  • We made a culture to make them competent toworrow (06.08)

06-08-2010

Wiki

Other

  • 11h32 Solange lives home

Biobrick

  • We obtain colonnies on the plate from yesterday. That from we maje miniprep

Asaia