Team:EPF Lausanne/Notebook

From 2010.igem.org

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== BioBricks ==
== BioBricks ==
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Ligation/Transformation C3+Promotor(weak/strong)+RBS
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Ligation/Transformation C3+Promotor(w/s)+RBS+Immuno
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Revision as of 17:19, 13 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

  • Made some plates 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.


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.

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

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 + i51020
  • 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

Biobrick

  • We obtained colonies on the plate from yesterday (Asaia origin + Resistance). We made a miniprep from that and stored some on Glycerol in the -80°C freezer. With the same miniprep we run a gel. Result :

Asaia

  • Measured the OD of our Asaia cultures. They were 1.24 and 1.95. We diluted them again and then started the procedure to make competent cells. Finally we got 22 aliquots of 150ul.
  • Had fun with the liquid nitrogen left over...
  • Plated one aliquot on Gly without AB to make sure the now hopefully competent cells survived the procedures.
  • We made some stock of "asaia origine + Resistance (Kan, Cm, Amp) + BackBones Vector" in the -80°C fridge.
  • We've transformed Asaia with plasmid C3 + E-coli Origin + Resistance and the colonie didn't grow so we can say it out and loud :
E-coli Origin doesn't work in Asaia

06-08-2010

Biobrick

  • We obtained colonies on the plate from yesterday (Asaia origin + Resistance). We made a miniprep from that and stored some on Glycerol in the -80°C freezer. With the same miniprep we run a gel. Result :

Asaia

  • Measured the OD of our Asaia cultures. They were 1.24 and 1.95. We diluted them again and then started the procedure to make competent cells. Finally we got 22 aliquots of 150ul.
  • Had fun with the liquid nitrogen left over...
  • Plated one aliquot on Gly without AB to make sure the now hopefully competent cells survived the procedures.
  • We made some stock of "asaia origine + Resistance (Kan, Cm, Amp) + BackBones Vector" in the -80°C fridge.
  • We've transformed Asaia with plasmid C3 + E-coli Origin + Resistance and the colonie didn't grow so we can say it out and loud :
E-coli Origin doesn't work in Asaia

09-08-2010

Biobrick

Asaia

  • We made pre-culture and plates (on both Kan and Gly alone) of the recovered WT Asaia
  • Made PCR of the promotor + RBS and ran gel -> it worked

10-08-2010

Immunotoxine

We'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.

Biobrick

We try to begin to construc two new biobrick today but we made mistake. We'll start again tomorrow.



11-08-2010

Immunotoxine

We'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.

Asaia

To 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.

Biobricks

We 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.

Drosophile

Persistence 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

Drosophiles

Text

Biobrick

Digestion of Immunotoxine Other jobs failed and we discovered we have to restart many digestion/ligation

02-08-2010

Drosophiles

It rain a lot today

Asaia

Digestion/Ligation of "ORI + Resistance (Amp/Kan)" into Base Vectors

BioBricks

Ligation/Transformation C3+Promotor(weak/strong)+RBS Ligation/Transformation C3+Promotor(w/s)+RBS+Immuno

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