| Week Twelve
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Sub-cultures of ETpuz cells containing pAU3-45 were set up (no growth for pMS82 cells) - 300 ul of cells in ~ 10 ml LB(+ MgCl2) and 10 ul Apr, Chl and Kanamycin. They were grown for 4 hr in a shaking incubator at 37°C.
Melted and poured 2 x 100 ml SFM (+ 100 ul Ny per 100 ml).
Lawns were performed of single Streptomyces colonies.
Performed spore stocks of lawns. Filtered, concentrated and resuspended spores in glycerol.
Set up more conjugations - spun down ETpuz pAU3-45 cells at 13 000 rpm for 10 min. Poured off supernatant and added 10 ml LB. Resuspended cells. This spinning and supernatant removal was repeated. The cells were then resuspended in 1 ml LB broth. The culture was split into 2 - 500 ul each. A total of 100 ul of Streptomyces spores was added to 500 ul of 2 x YT broth. This was heat-shocked at 50°C for 10 min and was allowed to cool. A 500 ul aliquot of E. coli ETpuz pAU3-45 cells was added to the heat-shocked spores. This was spun briefly and most of the supernatant was poured off. The spores were then resuspended in the remaining liquid. A dilution series from concentrations of 10^-1 to 10^-4 of the spores in a total of 100 ul sterile water was performed. A 100 ul volume of each dilution was plated onto SFM + MgCl2.
Our focus of the experiments was to prepare more DNA of the confirmed biobricks and to reclone our fourth brick.
To prepare biobrick J04450+neo; plasmids 3b and AntGpneo were cut with restriction enzymes NdeI and PstI. The samples were ran on a agarose gel, but the band were unclear so the gel was not used further.
DNA from 3a and BBa_J04450 were transformed into α-select gold competent cells and spread on LB agar plates with added chlorophenicol antibiotic and incubated overnight at 37°C.