User:Isis Trenchard/Notebook/BioE44 Stuff/2010/03/24

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Details of the first part of the class

Day 2 (Thurs April 1)

  • give them cultures with color generator vectors flanked with BB prefix and suffix (no promoter). K274002 (purple, do 2), K274003(dark green), K274004 (light green), K274100 (red), K274200 (orange)
    • actually, I should clone purple and green generators into plasmid with no promoter (like into pSB1A2) before class starts (right now they're on DNA2.0 expression vectors)
  • do mini-prep on cultures
  • use nano-drop to measure concentration of DNA
  • plan digestion mix
  • set up digestion

Day 2.5 (Fri April 2, TAs)

  • take digests, heat inactivate and store
  • (sometime before tuesday)Digest pBAD and LacI vectors

Day 3 (Tues April 6)

  • Pour gel
  • Talk about inducible promoters, input/output relationships
    • What promoters are available
  • Load samples onto gel
  • Run gel
  • Cut bands out of gels
  • Use kit to extract DNA from gels
  • Talk about ligations
  • Set up ligation with extracted DNA and precut vector of their choice

Day 3.5 (Wed April 7, TAs)

  • put away ligations
  • TA's set up cultures for electrocomp. prep

Day 4 (Thurs April 8)

  • electrocompetent cell prep
  • transformation
  • plate

Day 4.5 (Fri April 9)

  • TA's put plates away

Day 4.5 (Mon April 12 - students)

  • set up cultures with different concentrations of inducer (have them calculate stuff ahead of time)

Day 5 (Tues April 13)

  • spin down cells
  • extract pigments
  • look at peak absorbance
  • data analysis

Other stuff left to order

  • tubing for bunsen burners and aspirators
  • cellophane discs for moss culture
  • dissecting microscope?
  • inverted microscope?
  • acetone
  • IPTG
  • arabinose
  • Restriction enzymes: EcoRI, XbaI, SpeI, PstI
  • T4 Ligase
  • enzyme coolers (a big one and a small one)

Pigment extraction protocol

  • from cambridge 2009
  • We grew transformed E.coli MG1655 in LB culture at 37°C for 24 hours and collected cell pellet by centrifugation. Acetone was added to the cell pellet and warmed at 50°C for 10 minutes, allowing carotenoids (lycopene or β-carotene) in the cells to dissolve. The acetone extracts were then put into MicroPlate Reader for a full photospectrum scan (wavelengths between 300nm and 800nm). The absorbance curves showed peaks characteristics of the respective carotenoids: at wavelength 474nm for lycopene, and 456nm for β-carotene.

Biobrick plasmids and promotors

  • LacI promotor (BBa_R0011) - inducible by IPTG
    • 2009 distribution - Kit plate 1 - 6G - pSB1A2
  • pBAD promotor (BBa_I0500) - inducible by arabinose
    • 2009 distribution - kit plate 1 - 14N - pSB2K3

Vio operon details

  • from Cambridge 2009

DNA2.0 very generously agreed to synthesize the entire operon for us, we designed it to include all the five genes, each preceded by a ribosome binding site, and flanked by the prefix and suffix.

This will be held under a repressible promoter on the pJexpress cloning cassette from DNA2.0. We codon optimised the operon for both E. coli and B. subtilis, and designed it to include restriction sites with complementary sticky ends around vioD and vioC. This allowed us to remove both genes easily to create more colours from the vio operon.

Plan

3/24

  • transform BBa_R0011, BBa_I0500, BBa_J04450 (has pSB1A2) into DH10B.

3/25

  • inoculate positive colonies

3/26

  • miniprep
  • make freezer stocks
  • digest
  • gel extract

3/28

  • ligate and transform 002, 003, 004 into pSB1A3

3/29

  • inoculate colonies for freezer stocks

3/30

  • make freezer stocks



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