IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/08/01
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Inoculated LB broth with E. coli cultures from a pAU3-45 (with 10 ul Apr) and pMS82 (with Hyg in LB -NaCl) plate respectively.
Spore stocks were performed on more lawned plates that had sufficient colony plate coverage.
Sub-cultures of E. coli were set up to grow for conjugations with Streptomyces spore stocks - 8 different ones prepared. A 300 ul volume of culture was added to 10 ml of LB broth + the relevant antibiotics - pMS82 with 10 ul Hyg and chloramphenicol (Chl) and pAU3-45 with 10 ul Apr and Chl.
Set up the conjugations - the E. coli pAU3-45 and pMS82 sub-cultures were spun down for 5 mins at 4000 rpm. The cells were washed and resuspended in 10 ml LB broth and spun down again. The cells were resuspended in 500 ul LB per 10 ml of sub-culture. The Streptomyces spore stocks were heat shocked - 500 ul of each (not filtered or purified). The stocks were incubated at 55 °C for 10 mins. There were 8 spore stocks and the S4 strain. The E. coli cultures + heat-shocked Streptomyces spore stocks were plated together on SFM with 100 mM MgCl2. The plates were incubated overnight at 30 °C.
The Biobrick AntAgp+Xba+Neo was digested by restriction enzymes Xba1 and Nde1. The samples were then analysed by gel electophoresis, but after imaging the results of the digestion were unclear. The protein over expression was continued by inoculating two new LB solutions (1a,1b) with 100μL from the overnight culture. 40μL of IPTG was added to sample 1b and both cultures were left to incubate at 25°C overnight.