= 02-08-2010 =
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== Other ==
It rain a lot today

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== Asaia ==
Today I speak with an asaia and she answer to me ^^

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== Drosophile ==
 
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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.

Asaia

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

BioBricks

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

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

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)

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)

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

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

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

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.

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 (Pseudomonas in 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 on the plates. We hope to get an idea of the persistency of Asaia in the gut of Drosophila.

Drosophila

• Same thing as yesterday afternoon, Making plates to get the CFU-count
• Plates of yesterday with Pseudomonas look promising, the cells grew and the dilution was there.

Biobrick

Digestion of Immunotoxine Other jobs failed and we discovered we have to restart many digestion/ligation Digestion C3+Promotor(weak/strong)+RBS Digestion C3+Promotor(w/s)+RBS+Immuno

Drosophilae

• Continued yesterday's experiment with plating, this time the 48h persistence.
• Unfortunately the plates from yesterday don't look good. We might have killed all the bacteria by washing the flies in ethanol for too long.

Asaia

• Prepared a wild type recovery pre-culture to obtain competent cells. We're repeating the procedure from two weeks ago, because what we obtained then didn't seem to be the wild type (it grew on plates with Kanamycin).

Biobrick

Digestion of Immunotoxine Other jobs failed and we discovered we have to restart many digestion/ligation Digestion C3+Promotor(weak/strong)+RBS Digestion C3+Promotor(w/s)+RBS+Immuno

BioBricks

Today we came to check our culture of E-coli transformed with the plasmid C3 + Promotor + RBS (+ Immunotoxine) but no plate have grown!!

Asaia

We also transformed E-coli 3.1 with the Base Vector (BBa_I51020) + Asaia Origin + Resistance.

Asaia

Today we just come and check our transformed cells of yesterday. We have colonies on two plates. Work continue tomorrow.

Asaia

• transferred pre-culture for competent wild-type into flasks, diluted 1:50

BioBrick

• Digestion / Ligation of C3 vector with either a strong or a weak promoter, with and without Immunotoxin

Asaia

• Made about 30 aliquots (150ul) of competent wt-cells and stored them at -80°C. Using liquid nitrogen is fun!

BioBrick

• Transformation of E.Coli DH5alpha with the previous day's ligations
• Running a gel to check if the Base Vector with ORI (asaia) and a resistance were fine, no concluant results
• Liquid cultures of the colonies (E.Coli transformed with Base Vector + ORI (Asaia) + Res. )

Biobricks

• Plasmid miniprep from the previous day's liquid culture
• Digestion and gel to check if the Base Vector + ORI + Res were rights -> didn't work
• Successful transformation -> Liquid cultures of the E.Coli DH5alpha colonies (C3 vector)
• Colony PCR (didn't work)
• Restarting the digestion/ligation of the Base Vector with the ORI (Asaia) and Amp or Kan resistance
• PCR of the p25 and p28 proteins
• Gel to check the PCR (failed)

Drosophilae

• We're redoing the same experiment as last week: starving the flies for 2-3hours, then transferring them to tubes with food with bacteria. This time we were more careful when drowning the flies in Ethanol (never more than 5 seconds, to make sure the bacteria in the gut survive). We also added a fourth strain, Ecc-15, to the experiment.
• We also did the 3h survivance test by plating the crushed drosophilae on gly and LB medium.

Biobricks

The plasmids we sent to sequencing came back positive. We have now 4 biobricks.

1. C3 + Asaia_origin + Kan
2. C3 + Asaia_origin + Amp
3. C3 + weak promoter + RBS
4. C3 + strong promoter + RBS

We also ran a gel with C3 + asaia origin. It looks good.

Asaia

Drosophilae

We restarted the experiment with the drosophila. This time, we crushed 15 flies and we washed them in ethanol for only 2 seconds (maximum). We also did the plating of the flies 3h after infection

Biobricks

We did a digestion and ligation of our 2 vectors C3 + S and C3 + W with the immunotoxin, P25 and P26. In the end, we should have 6 new biobricks.

Asaia

We are still not sure if Asaia "WT" is resistant to kanamycin or not. Maybe we will have to use another resistance in our biobricks, TetR for instance.

Drosophilae

We are doing the crushing and plating of the drosophilae that were infected 24h ago.

Other

It rain a lot today

Asaia

Liquide culture of WT Asaia. We took the cultures of Asaia (5ml each) made the 27th to see in which antibiotic Asaia grew: Asaia did not grow on Kan, GM (Gentamicin), Tet and did grow on Amp, Cm and medium without antibiotics. For Tet something did grow so we made the cultures withj Tet again.

Drosophile