Regulatory

Part:BBa_K1067007:Design

Designed by: Kristian Davidsen and Helen Cook   Group: iGEM13_DTU-Denmark   (2013-09-28)
Revision as of 13:01, 12 October 2013 by Helencook (Talk | contribs) (→‎Design Notes)


Tight pBAD with araC


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1182
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1182
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1182
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961


Design Notes

  1. Random promoter sequences were ordered matching the sequence CTGACGNNNNNNNNNNNNNNNNNNTAWWATNNNNA.
  2. USER cloning to add RFP downstream of promoter.
  3. Colonies were plated.
  4. After inspection under UV-light the non RFP containing colonies (via visual inspection) where picked.
  5. Plates were induced by spraying them with an 5% w/v aqueous arabinose solution.
  6. The plates were again inspected under UV-light and this time the most red florescent cells were picked.
  7. Colonies were grown in culture tubes and screened in parallel on the BioLector. Wells on BioLector plate were loaded with culture by transferring a toothpick from each overnight culture selected and into the wells of the plate. All wells were run in duplicate.
  8. All duplicate colonies were run twice -- once with arabinose added at t=0, and again without arabinose.
  9. The pBAD system BBa_K808000 was used as a reference.
This reverse primer sequence is incorporating randomized promoter sequences into the pBAD construct. The sequence is annotated with -10 and -35 consensus region. Note that I2-bindingsite is overlapping with the -35 region. Blue is the binding part of the primer, red is the USER made sticky ends. N=random, W=50% A and 50% T

Data analysis

  1. Data was collected from the Biolector, and analyzed using a series of R scripts written by Chris Workman (unpublished).
    • The maturation and degradation times for mCherry were both assumed to be 40 min.
    • The growth rate, mu, was estimated to be 1.28 (from an average of all wells on all plates) since we expect each strain to grow at the same rate.
    • A time window representing exponential growth was selected (between 4 and 8 hours).
  2. The RFP measurement for a constitutively expressed strain was used as a standard measure of growth. This is plotted on the x-axis in the detailed plots per colony below.
  3. Figures were plotted using R.

Source

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References