1. MARCO REFERENCIAL
1.3 OBJETIVOS
2.2.3 EL DELITO DE ROBO
2.2.3.3 EL DELITO DE ROBO EN EL CÓDIGO ORGÁNICO INTEGRAL
In the case of the mutant, the fragment containing the complete gene and flanking sequence (4.1 kb) was amplified and kinased as described in the methods (see fig 5.1 for region amplified and construction of the initial knockout plasmid). This blunt-ended fragment was then introduced into the pBluescriptKS vector at the EcoRV restriction site
located in the multiple cloning site. The ligation mixture was then transformed into E. coli
D H 5a cells with blue-white screening and restriction digest testing being done to determine the presence of the insert. Primers were then designed to remove 405bp of the sigC coding
sequence by inverse PCR and also incorporate a SnaBl restriction site in its place. The
purpose of the site was to allow the introduction of an antibiotic resistance marker to aid in selection if the unmarked construct gave rise to many revertants to wild type. In addition, the presence of a positive marker would act as proof as to the viability of a mutant. The mutant might be out competed by the wild type and the addition of a selectable marker might promote its presence. However, in the case of the sigC mutant, it was decided to
proceed initially with the unmarked construct.
The resulting PCR product was kinased, re-ligated and transformed into super-competent E.
coli X L l blue cells. The resulting construct was modified by site-directed mutagenesis to
remove 159bp of the lacZ gene and introduce a P a d site in its place. In addition to being
able to introduce a screening and counter-selection cassette through the restriction site, it was hoped that the removal of the lacZ gene would stop any kind of homologous
blaC R v 2 0 6 7 S ig C co b K cobM cobL m u t a n t f r a g m e n t R e m i n a n t o f lacZ p r e s e n t in p B l u e s c r i p t . pB luescript sIgC I n v e r s e P C R t o r e m o v e sigC . information pathway Intermediary metabolism conserved pB luescript AsigC I n v e r s e P C R t o r e m o v e r e m i n a n t o f p B l u e s c r i p t lacZ a n d i n s e r t P a d s i t e in p l a c e . pC ulterl I n t r o d u c t i o n o f S a d d i g e s t e d K a n ^ c a s s e t t e f r o m p U C 4 K a t Xhol s i t e . pCuiter2
Figure 5.1. This figure illustrates the making of the M. tuberculosis âisigC knockout plasmid construct prior to the addition of the lacZ / sacB marker cassette from pGoall? (Parish and Stoker, 2000). The above section shows the PCR fragment containing sigC
and flanking region that was PCR amplified and placed in pBluescriptKS. The arrows represent which way each ORF is read whist the black region between sigC and cobK
indicates that there is a small overlap in sequence between the two genes. Each region is colour-coded for function.
It had been shown previously that the presence of markers on opposite sides of the desired deletion led to an increase in frequency of the correct double cross-over identified (Hondalus et al, 2000). Therefore, a kanamycin resistance gene was excised from pUC4k
and digested with the restriction enzyme SaK and ligated to the unique site Xhol site in the
mutant construct.
The final step was the addition of the marker cassette (a lacZ / sacB construct) from
pGOALl? (Parish and Stoker, 2000). After this final step, the complete plasmid (pCulterS) was ready for transformation into M. tuberculosis H37Rv. The lacZ gene in the marker
cassette would enable a blue-white screen to be used to identify colonies that had initially taken up the mutant construct as possible single cross-overs (signified by blue colonies). The sacB gene was to be used as a negative screen in the determination of double cross
overs, whereby sucrose resistant colonies grown on plates containing sucrose would signify loss of the vector. Using X-Gal plates at this step and screening for white colonies could also be undertaken as a confirmation of the loss of the vector.
It had been stated that UV or alkaline treated plasmid DNA had a greater tendency to be incorporated by homologous recombination in M. tuberculosis than non-treated DNA
(Hinds et a/., 1999). For this reason, it was decided to use alkaline treated as well as
untreated DNA to transform M. tuberculosis cells.
In the transformation, 120|ig of both treated and untreated S N A P miniprep (Invitrogen) DNA was used to transform 400pl of M. tuberculosis H37Rv. From these transformations,
crossover events (blue colonies), with the plates being scored after four weeks. The selection procedure is shown in Fig 5.2.
Only one colony was obtained, which happened to be blue and was from the transformation done with untreated DNA. This sole colony was streaked out on a 7H11 plate without any antibiotic pressure to allow for the double crossover event to occur. After three weeks of growth, a loopful of bacteria was taken and dilutions of 1x10 % 1x10 ^ and 1x10 ^ were made. These dilutions were plated on 7H11 plates with 2% sucrose and X-GaF°° to select for loss of both sacB and lacZ genes (loss of the vector and, therefore, the double crossover
event).
Twenty Sue* white colonies were isolated from the Ix 10^ plate on the basis of the right phenotype. These colonies were then patch tested on 7H11 Kan^^ plates and screened for Kan^ clones, which indicated that the vector backbone had been lost. Of the twenty colonies isolated, six were found to have the Kan^ phenotype. Genomic DNA was isolated from these six colonies showing the right phenotype for testing by PCR amplification and Southern blot.
mutant vector with Flanking construct Kan Single cross overs P a d digested
lacZ / sacB marker
Ligate and Electroporate in M. tuberculosis
Select on Kan and X-Gal (Blue)
Streak out onto antibiotic free plates to allow second cross over to occur
Resuspend in medium with glass beads and vortex to break clumps
Serial dilution
I I I
White colonies =
potential double cross over (loss of lacZ) Plate on sucrose and X-Gal Streak white colonies onto both
+/- Kan plates DNA preparation and Southern analysis of Kan sensitive colonies
+Kan -Kan
Figure 5.2. This figure illustrates the procedure by which the unmarked sigma factor mutants attempted in this study were created. This methodology was based on the work of Parish and Stoker, (2000) in the creation of a tly plcABC mutant in M. tuberculosis by homologous recombination.
The primers for PCR were based on the flanking sequence of the sigC gene and from this it
was seen that one isolate had an amplified signal that was expected of a knockout (fig. 5.3). However, the same signal would also be given from a single crossover and, therefore, a Southern blot was done to confirm the knockout. Probes of around 400bp based on sequences outside the 5’ and 3’ regions initially amplified with sigC were made. Genomic
DNA from the six strains isolated as well as the wild type were digested with Xhol (there
was a Xhol site in the wild-type that was missing in the mutant allowing them to be easily
distinguished (Fig 5.4). In the case of the wild type strain, band sizes of 4.1 kb or 4.9 kb would be expected depending on which probe was used while the knockout strain would give a band of 8.6 kb regardless of the probe used. From the resulting blot it was seen that one of the six isolates was indeed a knockout strain. The mutation was later confirmed by repeating the Southern blot with a new probe of about 300 bp based on the region deleted from sigC. In this case DNA from the knockout and mutant were digested with EcoRV.
Whilst a band of 2.7 kb was expected when the wild-type was probed, no band was expected in the mutant. This Southern as shown in fig 5.4 confirmed that a deletion had been introduced into sigC in this strain.
" O jg Q. n o o wt SCO 14 15 16 17 18 19
Figure 5.3. This figure illustrates the PCR amplification of DNA isolated from 6 potential M. tuberculosis AsigC double crossover colonies. Primers were based on
sequence in the flanking genes of sigC which were present on the fragment used to create
the knockout construct. These primers amplified up a fragment of around 1 kb in the wild-type and around 700bp in a correct M. tuberculosis AsigC double crossover. Wild-
type DNA and DNA isolated from a single crossover were used as controls. From this figure, it can be seen that only one sample, 17, is correct by PCR. Interestingly, although it was expected for there to be two bands in the single crossover corresponding to wild- type and mutant, only one band, corresponding to that of the mutant, was seen.
Ko wt Ko wt
c
Ko wt 2 3 1 3 0 - 2 3 1 3 0 - 2 3 1 3 0 - 9416 - 1 9 4 1 6 - 9416 - 6557 - 6557 - 6557 - 4331 -m
4331 - 4331 - 2322 - 2322 - 2322 - 2077 - 2077 - 2077 -Figure 5.4. This figure illustrates the Southern blot undertaken to confirm that sample 17
was a M. tuberculosis AsigC strain. Three probes were used in this study. One probe (A)
was based on the 5 ’ region outside the PCR fragment that was part of the mutant construct, whilst a second probe (C) was based on the 3’ region outside the PCR fragment that was part of the mutant construct. Both these probes were used in blots involving
Xhol digested wild-type and strain 17 genomic DNA. In the case of wild-type sequence,
probe A would hybridise to give a 4.1 kb band, whilst probe C would give a 4.9 kb band. In the case of a double crossover for a AsigC strain, both probes would give a band of
size 8.6 kb. In addition, a third probe (B), based on the region deleted from sigC by
inverse PCR was also used to probe both samples of genomic DNA., which in this case was digested with EcoRW. Whilst a band of 2.7 kb was expected from wild-type DNA,
no band would be expected in a true M. tuberculosis AsigC strain. From the resulting
blots it can be seen that strain 17 was indeed a M. tuberculosis AsigC strain.