Team:UGent/Results
From 2013.igem.org
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<b> Assemble T7-ccdB plasmid inside CcdB-survival strains </b><br><br> | <b> Assemble T7-ccdB plasmid inside CcdB-survival strains </b><br><br> | ||
- | When assembling the plasmid containing T7-ccdB in cells resistant to CcdB, mutations as a result of stress are less likely to occur. | + | 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. |
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Revision as of 02:43, 4 October 2013
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.
Strains with ccdA-gfp constructThe 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.
CIChE
Recommendations for further research
Assemble T7-ccdB plasmid inside CcdB-survival strains
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