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To perform in silico analysis, we used Clone Manager. All plasmids were first implemented into the software, and then used to help with cloning simulation, restriction, PCR etc.

Plasmid containing construct for chromosomal evolution

pTGD ccdA Pmb1 BCD7 GFP CmFRT

This plasmid contains the ccdA antitoxin and the reporter gene GFP. The homologous regions are necessary to perform Chemically Inducible Chromosomal evolution (CIChE). The removal of the chloramphenicol resistance cassette between the FTR sites is done using the method of Datsenko & Wanner [PNAS 2000].

Plasmids containing toxin ccdB

All of these plasmids are constucted using biobrick-standard parts: the plasmid backbones pSB3T5, pSB4A5 and pSB6A1. The ccdB gene that encodes the toxin is controlled by T7 promotor.

pSB3T5 T7 ccdB

pSB4A5 T7 ccdB

pSB6A1 T7 ccdB

New part

pSB1C3 T7 ccdB