Conversores

y c f 24 was amplified from purified LE392 DNA using primers APKl and APK2. Following digestion with Bam HI it was cloned into pK 03 and selected at 30°C in the presence o f chloramphenicol. Recombinants were screened for the y c f 24 insert by Bam HI restriction. Spl was cloned into the unique restriction site Nru I and clones containing yc/" 24 Spl-pK 03 sorted by resistance to streptomycin. Restriction analysis and sequencing confirmed the correct construct had been made.

A knockout experiment with this new construct was carried out, as described in the previous section. This time recombination o f y c f 24 Spl-pK 03 into the genome

M 1 2 3 4 5 6 7 8 9 10

1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0

Figure 3.3.3

PCR of potential knockout clones from the y c f 24 knockout using DNA isolated from IN V aF' cells.

Lanes 1-14 and 16-20. 19 potential positive clones which grew on 5% sucrose but not 5% sucrose + plates. All show a product o f l.Skb (arrows) corresponding to the size o f endogenous 24.

Lane 15. Negative control.

w M M H ^ r-* H M

1 2 3 4 5 6 7 8 9

Figure 3.3.4

PCR of clones resistant to sucrose + streptomycin and sucrose + chloramphenicol using prim ers designed to vector sequence, either side of the y c f 24Spl cloning site.

Lanes 1-9. 9 clones found to grow on both streptomycin and chloram phenicol in the presence o f 5% sucrose. All show a product o f ~3kb corresponding to y c f 24S pl.

was confirmed in four out o f a sample o f five large colonies grown at 42°C. This was done using primers spanning 24 and it’s two flanking genes (A PK l6 and A P K l7) in a long PCR reaction using LA Taq. Although no “long” products were obtained, in one reaction a product was observed which was the equivalent in size o f WT y c f 24 and it’s flanking genes i.e. no recombination having occurred (2.6kb as shown in Figure 3.3.5 lane 8). In the other samples, with the same reaction mix and approximate amounts o f DNA, no products were amplified suggesting that recombination o f the disrupted gene had occurred at the y c f 24 locus and prevented amplification o f this product.

Six large colonies were selected at 42°C and serially diluted onto 5% sucrose and 5% sucrose + streptomycin plates. In all cases colonies were observed under both of these conditions but the number o f colonies growing on streptomycin increased to approximately to one twentieth of those on sucrose only (Table 3.3.2). Again all colonies growing on 5% sucrose were large, whereas the colony sizes on plates containing 5% sucrose + streptomycin were much more variable. In addition to the occasional colony as large as those found on 5% sucrose (often only one or two on the plates with the most concentrated inoculum) most colonies were very small. Often these plates required two days growth at 30°C before they could be replica plated onto 5% sucrose + chloramphenicol. The very small colonies were assumed to be emerging sacB mutants as the majority were subsequently found to be resistant to chloramphenicol as well as streptomycin in the presence o f 5% sucrose.

As described previously, all clones growing on both 5% sucrose and 5% sucrose + streptomycin were replica plated onto 5% sucrose + chloramphenicol to screen for mutations in sacB resulting in the retention of pK 03. Again clones selected on 5% sucrose only rarely grew up under chloramphenicol selection (i.e. the vector had been lost) and screening o f 175 o f these by PCR indicated the presence o f WT y c f 24 and not the disrupted gene.

Unlike the previous knockouts experiment some clones (77) were found to grow in the presence o f sucrose and streptomycin but not in the presence o f sucrose and chloramphenicol. This indicated that y c f 24 had been replaced hy y c f 24Sp\ (III in Fig. 3.3.2D) and yc/’24-pK03 excised. This caused us to doubt that y c / 24 was an

1.3kb-

2.6kb

M

1 2 3 4 5 6 7 8 9 10

Figure 3.3.5

PCR to check for recombination of vector with insert sequence into the E. coli

genome.

Lanes 1-5. ndh control knockout. A PCR product o f approximately 1.3 kb, the same size as endogenous ndh, is observed in lane 3 indicating that no recom bination has occurred in this clone.

Lanes 6-10. y c f 2^ knockout. A PCR product o f approxim ately 2.6kb is marked in lane 8, the same size as 24 and it’s flanking genes. This again indicates that no recom bination has occurred in this clone.

Number o f Colonies

Dilution 5% sucrose 5% sucrose + streptomycin

1 1000 58 2 150 4 3 21 1 4 7 - 5 - - Table 3.3.2

Number of colonies observed after serial dilution onto 5% sucrose and 5% sucrose + streptomycin. Data from y c f 24 knockout using isogenic DNA isolated from LE392 cells.

essential gene in E. coli as originally indicated. However, PCR again showed that endogenous y c f 24 had not been replaced with the streptomycin disrupted version of y c flA (Fig. 3.3.6). Initially, to check that pK03 had been excised and lost, minipreps were carried out for a sample o f these clones resistant to sucrose + streptomycin but sensitive to sucrose + chloramphenicol. No plasmids were evident, confirming the loss o f pK 03. The resistance o f these clones to streptomycin indicated recombination o f either the streptomycin resistance gene or y c f 24 Spl elsewhere on the E. coli chromosome. To confirm this, PCR was carried out with a sample o f these clones using primers (A PK l2 and APKl 3) designed from sequence within yc/*24, either side o f the streptomycin resistance gene. The resulting multiple products included bands of the expected size for the WT (~160bp) and streptomycin resistance genes (Fig. 3.3.7A). Although this initially indicated recombination o f y c f 24 Spl had occurred elsewhere, multiple products o f the same sizes were also observed using WT genomic DNA as a template in the same PCR reaction (Fig. 3.3.7B). This made it difficult to determine exactly what had happened.

As the PCR approach described above failed to determine if the streptomycin resistant potential knockout clones contained disrupted y c f 24Spl, Southern blots were carried out using probes for both y c f 24 and aadA (Spl). Genomic DNA was isolated from four clones resistant to 5% sucrose + streptomycin but sensitive to 5% sucrose + cam. Three (31,32 and 35) originated from one 42°C clone, the fourth (50) originated from a second 42°C clone. Approximately 400ng o f each DNA sample was digested with Dra III and 200ng o f each was run in duplicate on an agarose gel. Following blotting, one half o f the nitrocellulose membrane was hybridized by WT y c f 24 and the other to the radio-labelled streptomycin resistance gene (Spl). As Dra

III cuts once within y c f 24 at position 1250 only two restriction products of approximately 4.8 and 2.2kb were expected in a Southern blot using WT y c f 24 as a probe. After hybridization with yc/"24, these were the only bands observed in all four clones (Fig. 3.3.8A). This indicated that following recombination, selection had favoured those bacteria carrying the streptomycin resistance gene recombined into the genome elsewhere than in the y c f 24 locus. Although it is not known where this recombination occurred, the Southern using Spl as a probe confirmed that the streptomycin resistance gene had recombined into the genome. Because as many as

? !- T p * 4 « » * * * « * # ^ f; # # * # # 1 # # 111# # *

M 1 2 8

Figure 3.3.6

PCR of potential positive clones from the y c f 24 knockout using DNA isolated from LE392.

Lanes 1-27. Clones found to be resistant to 5% sucrose + streptomycin but sensitive to 5% sucrose + chloramphenicol. All show PCR products o f l.Skb (arrow), the same size as y ç / 24 indicating that no knockout had survived.

Lane 28. Negative control.

A