User:Isis Trenchard/Notebook/BioE44 Stuff/2010/03/24: Difference between revisions

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*pBAD promotor (BBa_I0500) - inducible by arabinose
*pBAD promotor (BBa_I0500) - inducible by arabinose
** 2009 distribution - kit plate 1 - 14N - pSB2K3
** 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.


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Revision as of 17:02, 24 March 2010

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Reworking the first part of the class

  • realized that the way the first two weeks are planned right now, can't get everything done.
  • how to rework so students still learn cool stuff but will feasibly get things done?
  • need to order arsenic sensor
  • want students to think thru things and make decisions - not too hand-holdy

Version 2

Day 2 (Thurs April 1)

  • give them cultures with color generator vectors under inactive promotor? so no color expression under constituative promotor so they can see colors and start thinking about how to measure input/output, understanding behavior etc. Also read about and understand how their color generators work - give them parts numbers so they can explore the registry?
  • miniprep
  • nanodrop
  • digest

Day 2.5 (Fri April 2, TAs)

  • take digests, heat inactivate and store

Day 3 (Tues April 6)

  • make gel
  • load digest
  • run gel
  • cut bands, extract dna from gel
  • set up ligation (vectors already prepped by TAs - can choose type of inducible? either arabinose or iptg? high copy/low copy...)

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 full absorbance spectrum
  • data analysis

Other stuff left to order

  • tubing for bunsen burners and aspirators
  • cellophane discs for moss culture
  • dissecting microscope?
  • inverted microscope?
  • acetone

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.


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