IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/08/19

Week Twelve

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Floor One Performed spore titres - selected 15 different looking colonies. First added glycerol to each spore stock to make up to ~ 100 ul. Diluted each colony from 10^-1 up to 10^-10. The first dilution was 10 ul of spores in 90 ul sterile water (SW) followed by the second dilution of this total 100 ul in 900 ul of SW. Spots of 10 ul of each dilution from 10^-2 to 10^-10 were plated onto LB agar (all dilutions of a spore stock on one plate). The dilutions were actually 10^-4 to 10^-12, because taking 10ul to plate is another dilution in itself. A minimum of approx. 10^-8 spores is required for the Bioassay with Candida albicans. Incubated at 30 ° for 5 days. Gathered 73 spore stocks in preparation for the Bioassays. Prepared the plates for the Bioassays - spotted 2 x 5 ul of undiluted and unquantified spore stock (resuspended in glycerol) as mentioned in Barke et al (2010) onto SFM agar (without Nystatin as this would inhibit Candida albicans growth). Incubated at 30 ° for 7 days. Reference: Barke J., Seipke R.F., Grüschow S., Heavens D., Drou N., Bibb M.J., Goss R.J.M., Yu D.W., Hutchings M.I. (2010). A mixed community of actinomycetes produce multiple antibiotics for the fungus farming ant Acromyrmex octospinosus. BMC biology 8, 109. Floor Two The transformation plate fig 1, from Friday, containing BL21 pETAntA competent cells with plasmid AntGp-RFP had four colonies growing.. Fig 1: Transformation of plasmid AntGp-RFP into BL21 E.coli cells containing pETAntA plasmid and spread onto an LB Agar plate with Kanamycin and Chlorophenicol antibiotics added. Outreach The posters and banner for the forum event were sent for printing today.