"
Team:Groningen/protocol
From 2012.igem.org
Our team has developed a new plasmid backbone for expression in B. subtilis (BBa_K818000) by putting a double terminator (BBa_B015) upstream the suffix region. This terminator was inserted by using HindIII and EcoRI restriction site. General cloning protocol was used for the cloning of the device construct into the plasmid backbone. The reporter genes were inserted into plasmid backbone by utilizing the EcoRI and PstI restriction site, while the reporter genes were inserted later into plasmid backbone containing reporter gene using EcoRI and SpeI. Molarity ratio of reporter:plasmid = 3:1 was used in every ligation step. The ligation was done for 2 hours in room temperature before it was introduced into E. coli DH5α.
B. subtilis was grown overnight in Spizizen’s minimal medium (SMM) in 37oC. In the next day, 1 ml of overnight culture was transferred into 10 ml fresh SMM and incubated for 3 hours in a 37oC 220rpm. The starvation medium was added into the B. subtilis culture and incubated for another 2 hours. 300 µl of B. subtilis was transferred into 2 ml microcentrifuge tube, 10 µl DNA was added, and incubated in a 37oC 220rpm for 1 hour. B. subtilis transformants were inoculated in the LB plates containing certain antibiotic as a selection marker. Chloramphenicol (10µg/ml) was used as a selection marker for B. subtiliscontaining BBa_K818000 and BBa_K823023 plasmid backbone.
Starch agar plate was used for characterizing plasmid backbone that contains integration site on the amyE gene. This medium was made by mixing LB agar with 0.4% starch. After the B. subtilis grown overnight on this agar plate, iodine solution was poured over this medium. In 15 minutes the iodine can be separated from the medium (poured back into the bottle) and the clear zone around B. subtiliswildtype strain would be detectable by eyes.
Growth experiment is the setup we used to grow B. subtilis with the volatiles of fresh and rotted meat. Rotted meat was acquired by rotting around 20 gr of minced meat in a closed 100ml jar at room temperature for a day. Using a peristaltic pump an air flow was creates that flows over the meat and into the culture (see picture for setup). A stir flow with a magnetic stirrer was used to shake the culture during growth and the whole setup was placed in the 37°C climate room. We kept the fresh meat fresh by placing the jars on ice, the ensure similar results we also placed the rotted meat on ice. For the micro array an overnight culture was diluted to an OD600 of around 0.3 and left to grow to an OD600 of around 0.8. The cells were now in exponential growth phase and the medium in the growth setup was inoculated to an OD600 of around 0.1. After 2 hours growth with the meat volatiles the culture reached an OD600 of around 0.6 which is ideal for the microarray experiments.
For the pigment characterization assay we inoculated the medium in the setup with an overnight culture of B. subtilis with the desired construct at an OD600 of around 0.02. This was left to grow for 12 hours and samples were took every hour. The pigment is measured at 395 nm, and using a formula containing the OD600 the pigment concentration is measured. We also did a visual observation of the pellet.
Our sponsors:
