Team:KU Leuven/Research/Results
Results
Double Knockouts
The Centre of Microbial and Plant Genetics (KU Leuven) provided us with three E.coli K-12 strains, with each one representing the knockout for the genes tar, tsr or cheZ. The kanamycin cassette of the tar knock-out strain was removed by the enzyme flippase on pCP20. This excision was checked by PCR. Here the original knock-out strain of tar was used as a positive control and a band at 1232 base pairs is expected. If the cassette is removed, a band at 438 base pairs will be visible. Hereby, ten colonies were tested and all have lost their cassette (Figure 1).
Figure 1
Excision of the kanamycin cassette of the knock-out of tar.
As a positive control, the original knock-out of tar was used which should show a band at 1232 basepairs. If the kanamycin cassette is removed, a band at 438 bp will be visible. Besides, a 1 kb Plus DNA ladder of GeneRuler was used.
The PCP20 plasmid contains a temperature sensitive origin of replication. To remove this plasmid, the colonies were grown overnight at 42°C. PCP20 is resistant to ampicillin, this characteristic is useful to verify the removal of the plasmid. Single colonies were streaked on one LB plate with and one without ampicillin. Figure 2 proves that the PCP20 plasmid is removed in all plasmids.
Figure 2
Test to prove that the ampicillin resistant PCP20 plasmid is removed. The left plate contains ampicillin, the right plate contains no antibiotic.
We kindly received lysate from Oscar Torres. Our donor strains (ΔcheZ and Δtsr) were infected with this lysate. In figure 3, the plaques as result of the infection is visible. Some of the plaques will contain DNA of ΔcheZ and Δtsr due the sloppy packaging mechanism of the phage P1.
Figure 3
Plaques after infection of our donor strains (ΔCheZ and ΔTsr).
Contact
Address: Celestijnenlaan 200G room 00.08 - 3001 Heverlee
Telephone: +32(0)16 32 73 19
Email: igem@chem.kuleuven.be
