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. The original knock-out strain of tar was used as a positive control and a band at 1232 bp is expected. If the cassette is removed, a band at 438 bp is visible. 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 2Test 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 FIXME!.
The lysate was plated out on LB plates as control. No colonies are visible in figure 4, this means that are lysate is not contaminated by cells.
Control to check if the lysate of ΔcheZ and Δtsr is not contaminated with cells.
The plaques of the acceptor strains were extracted and different amounts of lysate were used to infect our donor strain (Δtar). The result was plated out on kanamycin plates to select the right colonies (Figure 4).
Figure 5
Infection of the acceptor strain (Δtar) by lysate originating from donor strains (ΔcheZ and Δtsr)
Our Tar knock-out cells without the kanamycin cassette were also plated out on kanamycin as a control. In figure 5 is visible that there is no growth.
Figure 6
Tar knock-out cells without the kanamycin cassette were plated out on kanamycin plates as control.
Different colonies were screened to confirm the knock-out in cheZ. If the cassette is not there, a band should show at 863 bp. If the cassette is still there, there should be a band at 581 bp. As a positive control we used a knockout Tar strain which does not contain the kanamycin cassette anymore.
Figure 7
PCR to check that cheZ is knocked out in Δtar. The positive control is a knock-out in tar which lost the plasmid.
To confirm that the knock-out in tsr was successful, a PCR was performed. A knock-out in tsr should give a band at 1304 bp while the original gene should give a band at 1964 bp. As positive control, a knock-out in tar which lost the kanamycin cassette was used.
Figure 8
PCR to check that tsr is knocked out in Δtar. The positive control is a knock-out in tar which lost the plasmid.
Especially in the new constructed ΔtarΔcheZ, the chance exists that the knock-out in tar is undone due the sloppy packaging mechanism of the phage P1. Therefore, the knock-out in tar is checked again by PCR (see figure 9). The first positive control (c1) is a tar knock-out who has lost the kanamycin cassette. As a second control (c2), a cheZ knock-out strain was used. The gel of figure 8 shows that all our ΔtarΔtsr strains are ok, while only two of the ΔtarΔcheZ are still ok.
Figure 9
PCR to check the knock-out in tar after the P1 transduction
Finally, the all the genes of the operon containing cheZ were checked by PCR (see figure 10). As positive control the tar knock-out which does not contain the plasmid anymore was used, but each time with the primers corresponding to the checked gene. The negative control contains the mastermix but without template.
Figure 10
PCR to check the operon of ΔtarΔcheZ
Motility Test
The cheZ knock-outs are not able to swim anymore. Therefor we performed a phenotypical motility test.The result is visible in figure 7.
Figure 10
Motility test of our cheZ knock-out
AHL detection
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Leucine detection
The standard curve from 0 to 100 µM did not give a linear relationship. Our working method needs optimisation. Because the enzymes are from other organisms than mentioned in Kugimiya and Fukada (2015), it is possible that the enzymes have another efficiency and as a consequence need to have another ratio (substrates over enzyme). Additionally, we did not have the same equipment as described in the article: we had to manually pipet the luminol solution. This possibly means that the measurements have a delay.
Due to a lack of time, we couldn’t complete the plasmid assembly and therefore, we were not able to proceed the quantification of leucine.
In comparison to HPLC, the chosen method would be less time consuming without the need of specialized equipment.
Contact
Address: Celestijnenlaan 200G room 00.08 - 3001 Heverlee
Telephone: +32(0)16 32 73 19
Email: igem@chem.kuleuven.be
