IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/08/07
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Gels were viewed under UV light (using the GelDoc) 'fig 1'. We were able to confirm that the PCR was successful as primer dimer bands (approx. 100 bp) were visible at the bottom. However, there were only showing a small number of DNA bands, suggesting that not enough was extracted.
Made 6 x 500 ml SFM, autoclaved for 1 hr, added Ny (500 ul per 500 ml) and poured into plates. Five plates were set aside to streak the Streptomyces colonies that took part in conjugation and grew - 3 colonies harboured pAU3-45 so 10 ul Apr was added to SFM and 2 colonies contained pMS82 so 10 ul Hyg was added to SFM.
Diluted the new soil samples (61-76) from 10^-1 to 10^-4. Plated the dilutions 10^-2 to 10^-4 on SFM + Ny.
Streak purified all remaining contaminated samples.
Lawned single Streptomyces colony plates.
A challenge presented itself when we discovered that the neo gene (for kanamycin resistance) may need to be changed as the reporter gene. Streptomyces sp. (S4 for certain) express AntA transiently, only in one stage of growth (substrate mycelia growth after sporulation but not in aerial hyphae) and so does not harbour kan. resistance after that leading to the death of bacteria. There was talk of using RFP (red fluorescent protein) or GUS (β-glucoronidase) as alternative reporter genes.
A 15% SDS PAGE was run using the soluble and his-tag purified samples from the protein overexpression at 150V for 1 hour.
The flights for the European Jamboree was booked.