Design and Construction of a Double Inversion Recombination Switch for Heritable Sequential Genetic Memory - Genetic Engineering: Recent Developments in Applications - page 183

four PCR reactions (“Culture PCR”). Four primer sets were used: 337 and 339
(which checks for state 0); 337 and 1006 (checks for states 2 and 3); 339 and
APS1 (checks for states 1 and 4); and 338 and 340 (checks for state 5). They are
labeled as primer sets E, F, G and H, respectively (Tables 1 and 2). The culture
PCR was performed as follows. A PCR master mix (containing 2.5 µl 10×Taq
PCR buffer, 0.2 µl 25 mM dNTP's, 0.2 µl taq polymerase, 0.5 µl 20 uM prim-
ers in 20.6 µl nuclease free water) was mixed with 0.5 µl pelleted cells. The PCR
cycling parameters were 95°C for 2 minutes, 35 cycles of (95°C for 30 seconds,
55°C for 30 seconds, 72°C for 90 seconds), then 72°C for 2 minutes, finally, hold
at 4°C.
The PCR products were run on 1% agarose gels, and visualized by ethidium
bromide stain under UV light. Sometimes the bands were cut and gel extracted for
sequencing. The vector itself also was sequenced for state identification. Because
different copies of a multi-copy vector might harbor different inversion switch
states, which would make sequencing impossible, plasmids were mini-prepped
after the induction and transformed into E. coli to isolate individual plasmids for
sequencing.
Acknowledgements
APA would like to thank Tom Knight of M.I.T. for pointing out the amazing
Online Encyclopedia of Integer Sequences which allowed us to be lazy about
proofs.
Author contributions
Conceived and designed the experiments: TSH. Performed the experiments: TSH
SKL. Analyzed the data: TSH SKL JDK APA. Contributed reagents/materials/
analysis tools: TSH SKL JDK APA. Wrote the paper: TSH SKL JDK APA.
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Memory in microbes: quantifying history-dependent behavior in a bacterium.
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3. Gardner TS, Cantor CR, Collins JJ (2000) Construction of a genetic toggle
switch in Escherichia coli. Nature 403: 339-342.