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

1. Today we amplifie P25 and P28 plasmid. First time it fail but the second one succeed. So now we have the P25 and P28 plasmid. Next step is to insert it after the strong and the weak promotors.
2. We also transformed E.coli 3.1 with the BV + Asaia origin + Amp resistance or Kan resistance.
3. With the same plasmid (BV + Asaia origin + Amp|Kan), we made a digestion with E and P then a gel but nothing works =(
4. We make miniprep from liquid culture containing E-coli transformed with
   1. C3 + Strong promotors
   2. C3 + Weak promotors
   3. C3 + Strong promotors + Immunotoxine
   4. C3 + Weak promotors + Immunotoxine

Then we digeste them with NheI to test if plasmids are correct but bad news juste the #3 colony is correct. =(

1. Day conclusion : bad day!

Asaia

Drosophile

BioBricks

As the digestion of C3 + [Weak promotor|Strong Promotor] + [ (nothing) | Immunotoxine] haven't work, we decide to make a PCR with all these plamsid but the result was negative. We also transform our linear C3 in a circular C3

Asaia

Drosophile

BioBricks

Asaia

Drosophile

BioBricks

It rain a lot today

Asaia

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

Drosophile