July

10/07/12

Plate WT Bacillus on LB media.
Prepare TFB1 & TFBII solutions, sterilize and store at 4°C.
Store sterile 1.5 ml eppendorf tubes at -20 °C.
Store sterile falcon tubes at -4°C.

11/07/12

Filter TFB solutions
Incubate the preculture at 37°C overnight.

12/07/12

Incubate the preculture again because ir fell down

13/07/12

Follow the RbCl2 competent cell protocol.
Transform Bacillus with pSB4A5

16/07/12

Our Bacillus subtilis transformation with psB4A5 failed; the only colony that grew was not pink, then we thought it was contamination and that our cells were not competent.
To elucidate what was the reason for what the bacteria did not grow, we plated our competent cells on LB media and transformed again but this time using puc19.
We also replated the only colony onto selective media.
We prepared all the solutions needed to make Bacillus subtilis competent according to Cambridge 2008 protocol (https://2008.igem.org/Team:Cambridge/Bacillus_subtilis_transformation)

17/07/12

Competent cells are ok; they grew on LB media.
The replated colony grew but is not pink as it should be. Our hypothesis was that this part is for coli, so the expression in bacillus would be lower. To know if the bacillus were transformed or not, we extracted plasmid.

18/07/12

Today we ran a gel to see if Bacillus had the plasmid but it has not.

September

12/09/12

We’ve made competent B.subtilis using the Two step transformation procedure We transformed our plasmid in E.coli RecA + by heatshock [Escherichia coli MC1061 heat shock transformation protocol]

13/09/12

Today we extracted plasmid by the Birbom –Doly method from our transformation in recA+, then we transformed bacillus with this plasmid.

14/09/12

Results: Bacillus subtilis were successfully transformed with pSB1AK3.

18/09/12

We’ve made competent B.subtilis using the Two step transformation procedure We also made competent E. coli RecA+ with CaCl2 procedure.

19/09/12

We transformed RecA+ E.coli with 10 microliters [65.5 ng/microliter] of bBAK143079 plasmid.

20/09/12

As the transformation didn´t grow, we transformed again with a higher concentration of plasmid: 20 microliters.

21/09/12

One of the petri dishes has 5 colonies; we made liquid culture to extract plasmid.