User:Tk/Notebook/MF-xfm/2008/04/09

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Contents

Transformation results

  • E. coli transformation
    • plated 100 ul, got 450 transformants
    • no significant difference between 1 and 2 hour outgrowth
    • This is 50x higher than last time, and 1.8e5 transformants/ug, higher than Epicentre QC
    • unclear what was done differently
    • put on ice for a period before transformation
    • transformed at 2KV rather than at 2.5KV
    • possible difference in SOC
    • Note: (4/10) The cuvettes were 1 mm cuvettes, not 2 mm, so this was 20 KV/cm field, quite high

CIRCE element location

  • Idea: use HrcA control + circe element as control for recT
  • Circe elements upstream of hsp70 and lon
  • perhaps test with a YFP construct
  • also need a good constitutive promoter, perhaps from EF-Tu upstream


Part construction

  • YFP
  • CIRCE element
  • Constitutive promoter
  • recT
  • additional selection marker -- CAT e.g.

Electroporation buffer

  • Remake EPB, but with added glycerol
    • 8 mM HEPES pH 7.4
    • 280 mM sucrose (95.5 g/liter)
    • 12% glycerol
  • The thought is that by adding glycerol to EPB we can avoid having to add it after washing cells, and the cells will be acclimated to the glycerol concentrations, avoiding having to wait for flash freezing. This may improve cell survival over the -80 freeze/thaw cycle.

The streptomycin story on Lartigue07

  • I had asked during review for an explanation about the improvement in genome transplant efficiency that came with streptomycin treatment. No explanation was given in the published version.
  • Dan Gibson today said that the streptomycin was used to eliminate yeast contamination in mycoplasma cultures. It was accidental that they discovered its activity in improving transplantation efficiency.

Additional selection markers

  • the chloramphenicol acetylase gene (cat) would be a good marker
  • Hahn99 uses it in M. pneumoniae transposon insertion (Tn4001)
  • The cat gene comes from pC194 (Streptococcus)
    • Horinouchi82a has sequence information and analysis
    • The article in the database is pE194 sequence, same author, year
  • Note Byeon84 which describes post-transcriptional control of expression of cat in pC194
  • The construction in Hahn99 used a BamHI fragment from pC194 and also a SmaI fragment, both active
  • See also Kenri04 for fluorescent proteins and use of cat
  • Mailed Duncan Krause for info on the 5' UTR of the gene in pKV104


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