crtW knockout

To observe the effects caused by the knockout, it is only strictly necessary to remove the gene activity from a wild-typeM. xanthusstrain. However, attempts were made to generate knockouts in four of the strains used in this investigation; DK101, DK717, DK718 and UWM 303, though no carotenoids should be produced in the latter strain. They could then act as controls for each other generating comparable results. A number of results were possible following gene knockout. Firstly gene removal may lead to a clearly visible phenotypic effect, which would be primarily observed as a colour change. As the principal carotenoid is no longer being produced the intensity of colour may either decrease or increase. A completely different colour may even be produced, caused by alternative carotenoid creation. It is also possible that there may be no visible alteration in colour. In this case proof would require the chemical identification of the carotenoids produced. Any other physical cell alteration detected would imply that the gene does not function as originally expected in carotenogenesis. The knockout created may potentially have no observable phenotypic effect on the cell at all. This would suggest that the gene was not functioning as a CrtW protein. The final possible scenario is that crtWremoval gives rise to a lethal mutation. In this case no detectable colony growth would be observed following recombination, indicating thatcrtWwas an essential gene inM. xanthus.

To confirm homologous recombination has taken place between the KO construct and M. xanthus DNA (Figure 5.8), the primary selection criterion is resistance to kanamycin. If following electroporation with the KO construct the tested M. xanthus strains produce colonies on DCY Kan50 plates, then it is highly probable that kan has been incorporated into the genome. Through a PCR of the resistant colonies the extent of recombination can be determined. In the case of only a single crossover successfully occurring the resultant PCR should be positive for both kan and crtW presence. If a double crossover has occurred then the PCR will show positivekanbanding but nocrtWpresent (Figure 5.8).

Using the previously adopted protocol for electroporation, the procedure was carried out using the four strains and the KO construct with selection on kanamycin media. All initial attempts resulted in plate contamination and very few M. xanthus colonies for either UWM303 or DK101. A number of colonies were produced on the

DK717 and DK718 plates though, numbering between 30 and 40 on each. Of these 20 were selected in total from each strain and the colonies replated onto separate DCY Kan50 plates. Repeat of the electroporation procedure using the UWM303 and DK101 strains, resulted in similar colony numbers being observed, although slightly reduced, with 50-60 produced in total for each strain. 20 of each were again streaked onto further selective media to verify that the colonies produced were kanamycin resistant M. xanthus. All tested colonies did prove to be resistant M. xanthus, although they grew in two distinct different colourations in the case of the DK101 strain; tan or a bright yellow. This phenomenon has been previously recorded (Kuner and Kaiser, 1981), and it is believed that the colour phase variance may influence spore development (Laue and Gill, 1995). It is clear this variance has no effect on non-sporulating colonies (personal communication, D. A. Hodgson), but both tan and yellow colonies were still sampled if they were present.

As every colony produced was kanamycin resistant, the PCR was carried out on all of the strains to verify whether the plasmid was present or flanking region crossover had occurred. Two novel primer pairs were designed that encompassed a region either end ofkanand then a small region from theM. xanthusgenome flanking regions. Following a PCR for each colony, none resulted in the production of agarose gel banding after electrophoresis. A separate check for kan gene presence using the original primers designed for amplification of kan from pDAH101 was conducted, and each gave a positive result. This confirms that all the colonies tested contained kanthat was being successfully expressed and all cells were kanR. The samples were additionally tested for plasmid presence using the ‘KO primers’ originally used during the creation of the KO vector, and all the returned results were negative. If any crossover is occurring, then it is likely that plasmid and genome intermediates would exist, and be detectable at this stage (Fig 5.8). As an additional control the samples were all subjected to further PCR to establish whether crtW was still encoded, each producing a band following gel electrophoresis. The results indicate that there has been no removal of the crtW gene, but all generated colonies following electroporation are still kanamycin resistant. Despite repetition of the experiment identical results were once again observed.

Following these results a range of additional PCR controls were carried out on the original isolated colonies using existing primers and primer pairs including: ‘DOU CHECK F’ with ‘KANR REV’ and ‘3rd REV’; ‘3rd REV’ with ‘3rd FOR’;

‘KANRFOR’ with ‘3rdREV’; ‘KANRFOR’ with ‘4thREV’ (Figure 5.9 and ‘Primers’ Methods). The only visible bands obtained after gel electrophoresis were for sequences normally present inMyxococcus xanthusgenomic DNA and none from the inserted plasmid, with the exception of thekan gene alone. Comparison of a control DCY Kan50plate of electroporated unaltered DK101 cells to a plate of an identical strain electroporated with the KO construct showed the latter to have three times as many resistant colonies growing. This suggests the results are not due to spontaneous kanamycin resitance alone. A second identical KO generation was attempted for the final gene in thecrtWoperon,mmb, which also proved to be unsuccessful.

Figure 5.8The three possible crossover events following transformation ofM. xanthusstrains with the KO construct PDP kan ORD KO vector pdp crtW ord mmb M. xanthus

X

kanPCR +ve crtWPCR +ve

KanR Single crossover atpdp Double crossover pdp kan ord mmb kanPCR +ve crtWPCR–ve KanR PDP kan ord mmb ORD crtW pdp

crtWPCR +ve kanPCR +ve

KanR Single crossover

Figure 5.9The location of the primers used to determine whether a double-crossover had occurred between M. xanthus genomic DNA and the KO construct. The primer sequences can be found in ‘Materials and Methods’.