User:Jeffrey Kim/Notebook/Halogenated natural products from uncultured bacteria/2009/01/13

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BB16 heterologous expression

  • Streptomyces Albus BB16 exconjugant spore stocks were grown up at 30C in 125 ml of SMM. 25ml were used in an EtAc extraction 1:1, 95:5 MeOH H2O extraction 1:1 (~47.5%), and an aqueous 25 ml supernatant concentration.
  • TLC analysis of EtAC extracts dried down and MeOH resuspended did not show appreciable differences in profile. NOTE: Culture supernatant N=2 and cell pellet were different color and composition from 436 transformed controls...something is in there.
  • EtAC extracts reuspended in 150ul of MeOH and 25ul injected over the LC/MS with JB 20 min gradient of AceNt 80 => 0 with MeOH blank. No notable differences in ESI - + or API - + profiles.
  • HILIC analysis of aqueous extracts will be performed on 50ul. (NOTE: very viscous so I may have to modify the proc a bit...NChemBio analysis of PPT just concentrated super down, may partition/filter first and then play around)

TAR cloning

  • Competent yeast innoculated for 5hrs with 1:100 dilution of overnight culture. Spun down at 1200xg for 5 minutes, washed with 40ml sterile dd H2O, spun down again, and resuspended in TFBI (250ul). 200ug of Herring Sperm DNA ss (boil 10m, ice 5m) added along with 200ng of US and DS pcr product, 600ng pLLX8 amplified cassette to 200ul of competent yeast in addition to TFBII (PEG solution). 30C for 30' and 42C for 15' before spinning down at 1200xg again. Pellet resuspended in 500ul TE and 200ul plated on uracil Dropout plates
  • O/N grown at 30C of the reaction mix and LLX13 vector alone as a control indicated a high degree of background (NheI digest of LLX13 was performed without BSE accidentally...could have reduced the efficiency quite a bit).
  • After 30h at 30C, full yeast colonies were swabbed with 5ml PBS, pelleted and swab prepped using a qiagen miniprep kit with the addition of vigorous vortexing with acid washed (500um) glass beads in the P1 resuspension step.
  • Final elution into 25ul of EB (1' settle) yielded [30ng/ul] DNA from both preps.
  • 2ul of the elution were electroporated into high efficiency EC100's (control and recombined plasmid) and allowed to outgrow for 1hr. Plated 1:10 and full reaction onto fresh LB plates with 100ug/ml AMP and 5ug/ml Tetracycline for selection of recombined plasmid