3.3 Knockout of-E c o /ijç /‘
3.3.3 Knockout using the temperature sensitive suicide vector pK
The key elements o f pK 03 include a temperature sensitive pSClOl {reoA) origin o f replication, the chloramphenicol resistance gene {cat^ for positive selection
and the sacB (levansucrase) gene for negative selection in 5% sucrose (Link et a l 1997).
y c f 24 Spl from pBSKS+ was amplified using the primers APKl and APK2 and cloned into the Bam HI site o f pK03. Following transformation o f E. coli strain IN V aF' by heat shock, transformants were selected at the permissive temperature, 30°C, on plates supplemented with 20 pg/ml o f chloramphenicol and 50pg/ml of streptomycin.
Following analysis by restriction digestion and sequencing, one recombinant plasmid clone o f y c f 24 Spl-pK 03 was used to transform LE392. As the frequency of recombination tends to be low, 10'^ to 10'^ (Link et a l, 1997), transformants were initially selected for growth at 30°C on both chloramphenicol and streptomycin. This ensured all clones contained yc/‘24Spl before selecting for integration o f y c f 24Sp- pK 03 into the genome. Integrants were selected by serially diluting four colonies from the 30°C plates onto pre-warmed plates at 42°C. At this non permissive temperature, circular pK03 episomes are unable to replicate due to the temperature sensitive origin o f replication, repA. Consequently, strong expression o f any antibiotic resistance genes carried by the vector (in this case cat and Spl) can only occur if a single crossover event has taken place to incorporate the vector into the host genome. Expression o f the genes carried on the vector are then under the control o f the host. This temperature therefore selected for homologous recombination between the endogenous copy o f y c f 24 and disrupted y c f 24 carried on pK 03, resulting in incorporation o f y c f 24 Spl-pK 03 into the host genome as an imperfect tandem duplication, and strong expression o f Spl and cat. In practise the temperature sensitive origin o f replication was not absolute, resulting in “slow growth” as opposed to “no growth” at 42°C. Therefore, to ensure clones were integrants only large colonies were used for the next stage o f the experiment.
Four large colonies growing at 42°C were picked and serially diluted onto plates supplemented with 5% sucrose and 5% sucrose + streptomycin before incubating overnight at the permissive temperature o f 30°C. As the expression of sacB carried on pK03 is lethal to E. coli in the presence o f sucrose, these growth conditions selected for a second crossover event (at point 1 or 2, Figure 3.3.2C), allowing replacement o f the endogenous copy o f y c f 24 and excision o f the integrated plasmid. In theory, growth on sucrose only selected for replacement o f the wild type
Figure 3.3.2
K nockout of E, coli y c f 24 using the tem perature sensitive, gene replacem ent vector p K 0 3 .
(Figure adapted from Link et al., 1997)
A. LE392 transformed with y c f 24Spl-pK 03 and selected on streptomycin and chloramphenicol at the permissive temperature, 30°C.
Result: Cells carry circular plasmid LE392 expressing Spl and cat. A small number o f integrants are likely but not selected for.
B. Serial dilution o f 30°C clones onto plates supplemented with both streptomycin and chloramphenicol at the non-permissive temperature, 42°C. As the circular episome is unable to replicate, streptomycin and chloramphenicol resistance is maintained by integrated plasmids.
Result: Single crossover event resulting in the integration o f 24Sp 1 -pK03 at the y c f 24 locus (expression o f Spl and cat under host control). Because the replication
control o f repA is not absolute, clones with integrated plasmid are larger than those without.
C. Serial dilution o f large 42°C colonies onto 5% sucrose and 5% sucrose + streptomycin at 30°C.
Result: Expression o f sacB renders pK 03 lethal in the presence o f 5% sucrose. Growth on sucrose therefore selects for a second crossover event (at point 1 or 2) which results in further recombination at ihQ y c f 24 locus and excision/loss o f pK03. Clones resistant to sucrose are potential positives and resistance to sucrose and streptomycin selects specifically for recombination o f y c f 24Spl. However, recombination resulting in replacement o f y c f 24 with y t/2 4 S p l at the y c f 24 locus can not be confirmed until the clones are screened for their sensitivity to chloramphenicol.
D. Potential positives are replica plated onto plates supplemented with 5% sucrose and chloramphenicol because mutations in sacB would allow retention o f circular plasmid in the presence o f sucrose following excisiop frçm the E. coli genome. Result: Clones resistant to sucrose + chloram phenicol contain circular plasmid. However, replacement o i y c f 24 withyc/*24Spl may have occurred. The two possible outcomes (shown in I and II) can be distinguished by
Clones sensitive to sucrose + chloram phenicol are potential knockouts. Clones found to be resistant to sucrose but sensitive to sucrose + chloramphenicol should contain either outcome III or IV (PCR will confirm which). Resistance to sucrose + streptomycin and sensitivity to sucrose + chloramphenicol however, selects for knockouts (homologous recombination at the y c f 24 locus resulting in its replacement with y c f 24 Spl) as shown in III. PCR must be carried out to confirm this, as recombination o f Spl can also occur at non-homologous sites.
repA (ts) sacB y c f 24Spl-pK03 y c f 24Spl c a t M13on
/A
y/
B /A
1
I
42°C streptomycin + chloramphenicol 7 /30°C 5% sucrose or 5% sucrose + streptomycin
/A
7 /
I
All potential “positives” D Replica plate onto 5% sucrose + chloramphenicole or / Sucrose or \ • streps y sucrose + strep'" \ + cm^ ^ sucrose + cm^ ^ Sucrose or sucrose + sucrose y c f 24Spl-pK03 y c f 24-pK03 PCR to confirm
gene with either y c f 24 Spl or a recombinant wild type gene (III or IV, Figure 3.3.2D), whereas selection on sucrose and streptomycin sucrose selected for recombination o f the disrupted gene only (III, Fig 3.3.2). In practise, before colonies could be screened for recombination and replacement o f endogenous y c f 24 all colonies resistant to sucrose (i.e. those selected on 5% sucrose and 5% sucrose + streptomycin) bad to be screened for their sensitivity to chloramphenicol. This was necessary to exclude any mutations that had occurred in sacB which would make pK 03 non-lethal in the presence o f sucrose. Consequently all clones were replica plated onto 5% sucrose + chloramphenicol.
Two scenarios were observed with the four recombinant 42°C colonies used in this way. In two cases approximately 30 times more colonies were observed on the 5% sucrose plates than on the 5% sucrose + streptomycin plates. An example is shown in Table 3.3.1. All colonies observed on 5% sucrose were large, whereas on the 5% sucrose + streptomycin plates most of the colonies were small. In the remaining two experiments, colonies were only observed on the 5% sucrose plates and not on the 5% sucrose + streptomycin plates.
Replica plating showed that all colonies observed on the 5% sucrose + streptomycin plates also grew on 5% sucrose + chloramphenicol, indicating that pK03 had been retained in the cells. However, as discussed by Link et a l (1997), it was still unknown whether the activity of sacB had been directy compromised by an internal mutation or whether a secondary mutation had taken place in the E. coli genome which conferred sucrose resistance. In these instances, where clones were resistant to sucrose + streptomycin and sucrose + chloramphenicol, one o f two recombination events may have occurred. The first being recombination followed by excision o f yc/"24 Spl-pK 03 (I in Fig. 3.3.2D) and the second, excision o ï y c f l A - pK03 following recombination o f y c f 24 Spl-pK 03 (II in Fig. 3.3.2D). Clones selected on 5% sucrose in the absence o f streptomycin selection were rarely (1-2%) found to grow on 5% sucrose plates supplemented with chloramphenicol. This indicated that in the majority o f cases mutations in sacB did not occur and, after the second recombination event, yc/'24Spl-pK 03 was excised from the E. coli genome and lost (IV in Fig. 3.3.2D).
Approximately two hundred o f these potential “positive” clones which grew on sucrose but not sucrose and chloramphenicol, were screened by PCR using primers
Number o f Colonies
Dilution 5% sucrose 5% sucrose + streptomycin
1 300 10 2 24 1 3 6 - 4 2 - 5 1 - Table 3.3.1
Number of colonies observed after serial dilution onto 5% sucrose and 5% sucrose + streptomycin. Data from y c f 24 knockout using DNA isolated from INVaF' cells.
A20 and A21 designed from genomic DNA sequence either side o iy c flA . All PCR reactions resulted in a product the size of WT y c f 24 only (Figure 3.3.3). These results indicated that as no knockout had been achieved, y c f 24 is an essential gene in E. coli. It remained however, to prove that no recombination events had occurred.
Although the clones resistant to 5% sucrose + streptreptomycin and 5% sucrose + chloramphenicol were found to harbour pK 03, recombination events may still have occurred at the endogenous site o f y c f 24 as mentioned above. Brown et al. (1995) showed that the essential gene, murA^ could be replaced on the E.coli chromosome as long as another copy was present to complement it. However, PCR using primers designed to genomic sequence on either side o f the recombination site amplified only the WT gene. Furthermore, PCR using primers either side o f the cloning site within pK 03 (pK03-L and pK03-R) indicated the presence of y c f 24 Spl-pK 03 (Fig. 3.3.4). This confirmed that replacement with the streptomycin resistance gene had not occurred at theyc^24 locus (I as opposed to II in Fig. 3.3.2D).
The failure to “knock-out” y c f 24 as shown in the results so far, appeared to indicate that y c f 24 was an essential gene (personal communication, George Church, Harvard, USA). However, no recombination events had yet been observed directly. One possible explanation for the failure was that mis-matches had prevented integration, as the y c f 24 gene used for the knockout was amplified from the INVaF' strain o f E. coli and not that of LE392. To exclude this possibility the knockout experiment was repeated using LE392 isogenic DNA. A parallel knockout experiment was also undertaken as a positive control using the non-essential E. coli gene ndh (Calhoun gr a/. 1993).