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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):

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:

UV Test

Phage transduction was used to perform the deletion of the recA gene after chromosomal evolution. To check whether the gene was successfully knocked out, an UV test was developed. The UV light puts a certain amount of stress on the bacterial cells. In respons to this stress, an SOS pathway is triggered in which recA plays an important role. Cells in which the recA gene is deleted will not survive on the UV-treated plate. Colonies in which recA was successfully deleted still can be found on a non-treated backup plate.

RESULT:

recA positive strain grows on both parts (UV and non-UV) recA negative strain 10’’ – no visible differences 15’’ – still some colonies on UV-part of the plate 20’’ – one colony visible on UV-part of the plate 30'' – no growth on UV-part of the plate

CONCLUSION
With a 30’’ UV exposure, the cells in which recA is successfully deleted will not survive on the UV-treated plate.

CIChE

Recommendations for further research

Assemble T7-ccdB plasmid inside CcdB-survival strains

When assembling the plasmid containing T7-ccdB in cells resistant to CcdB, mutations as a result of stress are less likely to occur at this stage. We experienced difficulties cloning the T7-ccdB construct in our plasmids probably because of leaky expression of the T7-promoter.