Team:DTU-Denmark/pBAD SPL

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(Experimental procedure)
(Experimental procedure)
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# The constitutive promoter TODO:which was used as a reference.
# The constitutive promoter TODO:which was used as a reference.
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[[File:AraSPL primer.png|thumb|upright=3.5|center|alt=Primer for pBAD SPL|Reverse primer sequence for 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]]
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[[File:AraSPL primer.png|thumb|upright=3.5|center|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 ===
=== Data analysis ===

Revision as of 09:45, 3 October 2013

pBAD SPL

Contents

pBAD synthetic promoter library

As a tool for expressing lethal proteins in E. coli we made a synthetic promoter library (SPL, RFC 63) with the pBAD arabinose inducible promoter. The concept was taken from the DTU iGEM team from 2010.

Methods

Experimental procedure

  1. Random promoter sequences were ordered matching the sequence CTGACGNNNNNNNNNNNNNNNNNNTAWWATNNNNA.
  2. USER cloning to add RFP downstream of promoter.
  3. Colonies were plated.
  4. Plates were induced by spraying them with an aqueous arabinose solution.
  5. Colonies that were not red prior to induction with arabinose but that did turn red after induction with arabinose were selected and re-innoculated as liquid cultures.
  6. Biolector: Wells were inoculated from overnight cultures of each of the selected colonies. All wells were run in duplicate.
  7. All duplicate colonies were run twice -- once with arabinose added at t=0, and again without arabinose.
  8. The constitutive promoter TODO:which 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. TODOref
    • 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 1 and 4.5 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.

Results

Summary

Promoter activity when induced (with arabinose added) plotted vs basal activity (without arabinose; ie leakiness of the promoter). The colonies that we selected all show less activity than the the constitutive promoter, and when induced, show higher activity than the constitutive promoter. Induced vs basal.png

Details

Promoter strengths for two trials of each colony with and without arabinose.

Colony Number With arabinose 1 With arabinose 2 Without arabinose 1 Without arabinose 2
Col28.90257.86990.7270.9552
Col39.272412.11420.52480.6982
Col49.457111.45220.52310.2508
Col126.364110.53890.58970.6869
Col107.96979.79490.33920.733
Col97.856320.10940.69950.7432
Col812.231815.45480.42030.4538
Col1311.03777.33430.4820.4641
Col1515.68178.27070.81690.1343
Col1814.791615.57360.66740.6745
Col1914.212616.48980.45450.3566
Col297.185316.34670.54450.5013
Col269.77249.62690.71180.7865
Col228.41685.59580.60490.5645
Col339.19828.99870.65081.374
Col3410.69877.8830.50670.5031
Col3513.84277.54690.43630.6281
ConRef6.5067.93238.78117.9323

Dtu-Fss-plot-col2.png Dtu-Fss-plot-col3.png Dtu-Fss-plot-col4.png Dtu-Fss-plot-col12.png Dtu-Fss-plot-col10.png Dtu-Fss-plot-col9.png Dtu-Fss-plot-col8.png Dtu-Fss-plot-col13.png Dtu-Fss-plot-col15.png Dtu-Fss-plot-col18.png Dtu-Fss-plot-col19.png Dtu-Fss-plot-col29.png Dut-Fss-plot-col26.png Dtu-Fss-plot-col22.png Dtu-Fss-plot-col33.png Dtu-Fss-plot-col34.png Dtu-Fss-plot-col25.png Dtu-Fss-plot-conref.png

Example of use

The tight inducible pBAD promoter was used in our "Hello World project" to regulate the expression of GFP SF, which was tagged with a signal peptide to direct it into the periplasm. Production and folding of GFP SF is faster than the transport system of E. coli, which leads to undesired accumulation of GFP SF in the cytoplasm. Only when using a promoter with low leakiness it is possible to translocate a significant fraction of GFP SF after its production has been switched off. Thereby we get a clear signal from the periplasm with low interference from the cytoplasm.

Alt text
Overview microscopic pciture showing "Hello World" transformants.
Alt text
High resolution picture of "Hello World" transformants, showing a clear seperation of green and red fluorescence. GFP is primarily located in the periplasm while RFP is located in the cytoplasm. Fluorescence intensity measurements are taken along the cross-section indicated by the white line. Picture taken with a confocal microscope and ??? filter.
Alt text
Graph of flourescence measurement (or is this the model???) on green and red channel. It can be seen that the intensity of the red flourescence is restricted to the cytoplasm while green flourescence has it's peaks on the egdes. A weak green signal is measured for the cytoplasmic region because the periplasm envelopes the cytoplasm.


See also "Hello World project".