Characterizing the SplC protease of
bovine-adapted Staphylococcus
aureus and observing any activity
against casein
9 different S. aureus strains were tested for casein
digestion and milk curdling after which I chose the two
strains: AR802 and RF122 for further experimentation.
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55 kDa
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An SDS-PAGE was performed with the following S aureus strains:
VI54661, VI54649, VI54665, VI54655, RF287, 283, RF122, 114,
AR802 . This was done to separate and identify the proteases via
comparison of the band size to the protein ladder.
The zymography technique with a gel consisting of casein was
then used to detect any proteolytic activity by proteases against
casein for each of the strains. The zones of clearance in the
casein gel were used to give an estimate of which S. aureus
proteases were able to digest casein.
As seen in the zymogram gel below, the RF122 produced big
zones of clearances in the zymogram, indicating large digestive
activity of casein. The two strains of choice were RF122 and
AR802 and hence were used for further investigation and the
cloning of SplC protease.
35 kDa
25kDa
70 kDa
55 kDa
40 kDa
35 kDa
25kDa
Preparing for DNA cloning
Initially an analysis of lineage-specific genes of SplC in both AR802 and RF122 was done which
helped in designing specific forward and reverse primers for two different plasmids.
 The two plasmids used in the cloning were pCT::vwb which was his-tagged (and lacking a STOP
codon) and pALC 2073 without a tag. Preparation for cloning was commenced by extracting
genomic DNA from the two strains AR802 and RF122 by lysing the bacteria samples with
lysozyme and lysostaphin. The Plasmids were then extracted and loaded onto agarose gel to
confirm plasmid DNA presence after which they were extracted from the gel??.
Next, a PCR was performed of the mastermix containing the extracted gDNA, designed primers,
DNA polymerase and dNTPs. The PCR products were then purified. State WHY
Cloning
For 3-day faster cloning method, the Gibson assembly technique was
used to insert the purified PCR products into the vector plasmid digest.
The DNA fragments from the PCR product were assembled into the two
plasmids using a thermocycler. To produce large quantities of the
plasmids with the gene of interest, IM08B E. coli competent cells were
made and then transformed with the assembled fragments from the
Gibson assembly via heat shock. The cells containing the plasmid and
gene of interest were ampicillin resistant and therefore grew on
ampicillin antibiotic plates.
For additional verification of plasmid presence in E. coli, colony PCR was
done to screen the colonies containing the desired genetic construct and
viewed on agarose gel. The plasmid from the chosen colony was
extracted from overnight E. coli cultures and electro transformed them
into USA 300 Δ proteases S. aureus strain. The USA300 Δ proteases
electrocompetent cells containing the plasmid + SplC gene were
Chloramphenicol resistant and therefore had the ability to grow onto the
plates. To further confirm that the plasmids were successfully taken up
by the S. aureus samples, they were first lysed, and a colony PCR was
performed to be observed on agarose gel. Successful results can be seen
on the left.
Induction 24hrs
Induction 2hrs
Results
 Next, to identify whether the SplC protease was being expressed, the
anhydrotetracycline was added to the samples for a 24-hour induction before
performing an SDS-PAGE.
 From previous calculations, the predicted protein molecular weight for
SplC_AR802 was 22.5 kDa and 22.4 kDa for SplC_RF122. However as seen in
the figure on the left, no additional bands were displayed in the SDS-PAGE gel
for the transformed USA 300 strains compared to the plasmid pALC control
(did not contain SplC gene insert), and subsequently no zones of clearances
were formed in the zymogram either.
 It was possible that the SplC protein was being expressed at low levels. Hence,
I repeated the experiment a second time by reducing induction time to 2
hours instead of overnight and the samples were filter sterilised and
centrifuged. Both the concentrated supernatant and pellet were used test on
the SDS-PAGE gel. The third time I performed an ethanol precipitation of the
samples to precipitate the proteins before visualizing them on the SDS-PAGE
gels. Both these changes did not produce the required sized bands on the gel.
Ethanol Precipitation
Results and Conclusion
 Additionally, a milk curdling experiment was done by adding milk
into each sample and inspected for milk curdling after 24 hours.
However, the results presented no milk curdling from pALC::SplC
AR802/RF122 and pCT::SplC AR802/RF122.
 Lastly, I resorted to using a his-tag antibody to perform a western
blot in order to detect the his-tagged SplC protein from the
pCT::SplC AR802 and RF122 samples. The results did not reveal
any bands on the western blot for the his-tagged plasmid as seen
on the right.
 The results obtained from multiple tests concluded that the SplC
protein was not expressed by both AR802 and RF122 S. aureus
strains despite the strains having taken up the plasmid.
I would like to thank Professor Ross Fitzegerald for
providing me with this invaluable learning
experience in his laboratory as well as thank Dr.
Amy Pickering for her teaching and guidance
throughout the project!