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Team:Warsaw/Stage1/RBSMeas
From 2010.igem.org
RBS measurement
Experimental setup
We have measured following Ribosome Binding Sites from Anderson's collection: J61100 J61107 J61117 J61127. Additionally we have measured following parts from the Community collection: B0030 B0031 B0032 B0033 B0034
We have used J23100 as our reference promoter and E0040 GFP as a reporter. Example construct:
<partinfo>BBa_K299007 DeepComponents</partinfo>All measurements were conducted in E.coli Top10 chassis.
We have decided to use Fluorescence Activated Cell Sorter (aka. FACS) as our measurement device. This allowed us to achieve greater precision because we didn't have to determine both OD and fluorescence separately (accumulated error from 2 measurements). Also flow cytometry allowed us to measure large population of cells in single run. You can think about this as conducting 30 000 independent experiments at once.
We have used following protocol:
- Each experiment was started from the fresh transformation of Top10 cells
- Strains encoding our measurement constructs were seeded from single colony and cultured in LB broth overnight to reach saturation phase. Measurement of each RBS was preformed from three independent cultures.
- Overnight cultures were spun down, resuspended in equal amount of PBS supplemented with 500mg/ml kanamycin and measured on BD FacsCalibur flow cytometer.
The results that we obtained are summarized in the figure below. You can easily notice that all RBS sequences from the Anderson's library are rather weak. Our measurements of community collections are in agreement with previous experiments, except B0030. In our hands B0030 is stronger than previously measured. It was suppouse to be around 0.6 of B0034, we observed 0.9 of B0034. For detailed comparizon of our results with registry and predictions see modeling page.
We sequenced all measured parts and it contained no mutations, also we measured the RBSes independently 2 times, each time all constructs were measured in triplicate. We are really curios if B0030 is actually stronger that we thought or it was our particular measurement system or technique that influenced the results.
We would like to encourage other iGEM team to re-measure B0030!
