Plasmids containing T7-ccdB

We constructed plasmid pSB6A1-T7ccdB for use in CIChE. Through this plasmid, pressure can be put on the CIChE strains simply by adding IPTG.

We also purified plasmids p5SpFRT-T7ccdB, p10SpFRT-T7ccdB, p20SpFRT-T7ccdB from strains we obtained from Inbio. These plasmids have different copy numbers and they too can be used in chromosomal evolution.

Having plasmids with different copy numbers at our disposal, we can test CIChE with different degrees of toxin-pressure.

Strains with ccdA-gfp construct

The following strain containing the construct to be duplicated (ccdA-gfp) was constructed (KI experiment 1):

E. coli MG1655 ΔendA DE3 ccdA-Pmb1GFP-CmFRT (called strain 8)

We also dispose of a backup version of this strain, constructed by Inbio in case our own experiment failed (called MDM.Cm). We included this second version in our further experiments as to test possible differences between the two versions.

CIChE Strains

The plasmids and strains mentioned above were used in transformations (experiment 3) to obtain strains containing all elements necessary to perform chromosomal evolution. The following strains were constructed:
8+pSB6A1-T7ccdB
8+p5SpFRT-T7ccdB
8+p10SpFRT-T7ccdB
8+p20SpFRT-T7ccdB