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Alkaline Phosphatase: Assessing Streptomyces Griseus as a Model Organism
Santina Bleil ‘12 & Leah Frenette ’12
Department of Biology, Lake Forest College, Lake Forest, IL 60045
Abstract
II. Binding Site Determination
VI. Gel Electrophoresis
IV. Dilution and Cell Concentration
Hypophosphatasia is a disease resulting from decreased alkaline
phosphatase activity. Alkaline phosphatase activity is controlled by zinc
levels because of zinc’s catalytic effect upon binding to the enzyme.
Streptomyces Griseus was identified as a model organism based on a
BLAST and Zinc 1 binding sites located at nucleotides 326, 330, and
412 were targeted as possible areas of mutation that would cause
hypophosphatasia. Primers were designed around the binding sites and
PCR was performed. Gel electrophoresis confirmed the primers are
accurate, allowing for replication of this segment of DNA in future
studies. Experimental mutations of these sights in S.griseus compared
to known mutations causing hypophosphatasia in the human ALPL
gene will lead to more knowledge of the disease and possible
treatments.
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Introduction
1.  Streptomyces Griseus is a filamentous bacterium that is a well
known organism in the production of antibiotics.
S. griseus is
alkaliphillic meaning it lives in basic conditions, the prime
environment for alkaline phosphatase.5
2.  Hypoposphatasia is a genetic disease that is characterized by
demineralization of the bones and teeth.2 The disease is caused by a
mutation in the ALPL gene.1
Figure 3. Uniprot Analysis of the StrK gene by similarity. Zinc 1 and 2
binding sites were found using Uniprot. Zinc 1 was focused on because they were in
a similar region and would be easier to isolate. Zinc 1 binding site are determined to
be at 326, 330, and 412.
II. Streptomyces Griseus as a Model Organism
Dilution
1
2
Expecte
103
105 Cells/
d
Cells/75
75 µL
Concentr
µL
ation
3.  The ALPL gene is the gene coding for Alkaline phosphatase in the
human liver, bone and kidney tissue. 1 Mutation in the gene causes
a production of an abnormal version of alkaline phosphatase, that
cannot function properly, causing build up of substances normally
processed by the enzyme. 1
4.  Alkaline phosphatase is a zinc-containing and zinc-dependant
metallo-enzyme, meaning that in cases of zinc deficiency alkaline
phosphatase activity is severely decreased. 4
3
4
5
Control
102
Cells/75
µL
10 Cells/
75 µL
1 Cell/
75 µL
0 Cells/
75 µL
E. Coli
Colonies
per Plate
800-1000
30
6
2
0
0
S.
Griseus
Colonies
per plate
1
0
1
0
0
0
•  Alkaline Phosphatase activity in lobster hepatopancreas tissue
is greater than in heart tissue.
Figure 6. Dilutions of S. griseus versus E.coli to test for cell
concentration. Dilutions from an original stock of assumed concentration, 2x109
cells per 1000 µL, were made to dilute the stock to the given expected
concentrations. Based on dilution 2 for E.coli it was calculated that the original stock
was actually 2.25x109 cells per 1000 µL. This is in acceptable range of the predicted
value, assuring our methods and calculations were accurate. The S. griseus
colonies did not grow as expected, this is assumed to be caused by the tendency for
the cells to clump.
Found on Chromosome 1, base pairs 21,708,455 to 21,777,488.
I. Assessing Alkaline Phosphatase
Activity in Lobsters
Figure 4. A ClustalW alignment of the S. griseus StrK gene and
Human ALPL gene was performed. The ClustalW alignment of StrK, the
gene responsible for alkaline phosphatase in S. griseus and Human alkaline
phosphatase ALPL gene revealed high conservation of amino acids between the
two species. This section of the alignment is the region that contains the 3 zinc
binding sites that were further examined. The sites at 326 and 330 bp are
identical while the third site at 412 is not conserved. Having 2 sites out of 3
conserved leads to the conclusion that the S. griseus StrK is similar enough to the
Human ALPL gene to act as a model organism.
strongest band at ~ 396 bp, while there was also a dark band at ~1000 bp.
Our positive controls met their expected lengths. The negative control
showed no DNA.
Conclusions
5.  Alkaline phosphatase has two zinc binding sites, we focused on the
zinc 1 binding site while designing our primers. 3
Figure 1. Location of ALPL, Human Alkaline Phosphatase gene.
Figure 8. Gel Electrophoresis of the DNA acquired from PCR
with our experimental primers. Our experimental DNA showed the
•  S. griseus StrK gene and Human ALPL gene have many
conserved regions, including all but one zinc 1 binding site.
•  The secondary structure at the two conserved sites is a coil,
while the varying site is part of a β-sheet.
•  An original stock of E.coli was determined to have a
concentration of 2.25x109 cells/mL. It was not possible to
determine cell concentration of S. griseus because of its clumping
nature.
•  Based on our gel electrophoresis, our primers successfully
replicated our target region of DNA (~396 bp).
V. Designing Primers
•  The presence of the second band (at ~1000 bp) could be
caused by the similar primer sequence found by the BLAST
elsewhere in the genome.
•  S. griseus is a good model organism for examining alkaline
phosphatase.
Future Studies
III. Secondary Structure
•  Knowing that these primers work, we can isolate the target
region and make mutations at and around the zinc 1 binding
sites and introduce them to the S.griseus genome and
examine the effects.
•  We can compare our mutations to known mutations causing
hypophosphasia, as there are many that very in severity of
symptoms. 2
•  We can work to improve the purity of our PCR to eliminate
the unwanted bands.
Acknowledgements
Figure 2. Alkaline Phosphatase was found to be significantly more
active in hepatopancreas tissue versus heart tissue in lobsters.
The hepatopancreas consists of digestive tissue while the heart is a contractile
muscle. This is expected because Alkaline Phosphatase is known to be
prevalent in tissue that transport nutrients.
Figure 5. A PSIPRED analysis of the secondary structure of strK
was acquired. The Zinc 1 binding sites at 326 and 330 are shown to be
coils while the site at 412 is part of a β-sheet. This could possibly be a
reason for the differing sequences seen above. The coil arrangement
makes for easy interaction with the Zinc molecule compared to a helix
because it is more flexible and available.
Figure 7. Primers were determined around the zinc 1 binding sites.
This work was supported by Lake Forest College. We would also like to thank Karen Kirk, Will Press, Beth Herbert, and Saajidha
Risvydeen.
The expected amplification product encompassed 396 base pairs. The primers
were tested for self complementarities and were found to have none. A BLAST
was also performed to look for highly similar sequences in other regions of the
S.griseus genome, and it was found that one primer had a similarly matched
sequence, the other was sufficiently unique.
1. Genetic Home Reference. (2007). Retrieved April 6, 2010, from ALPL: http://ghr.nlm.nih.gov/gene=alpl
2. Genetics Home Reference. (2007). Retrieved April 6, 2010, from Hypophosphasia: http://ghr.nlm.nih.gov/
condition=hypophosphatasia
3. Kirk, K. (2010, Spring). Lab Handouts: weeks 1-15. BIO221: Cell and Molecular Biology . Lake Forest, IL, USA.
4. Naber, T. H., Baadenhuysen, H., & Jansen, J. B. (1996). Serum alkaline phosphatase activity during zinc deficiency and long term
inflamitory stress. Clinica Chemica Acta , 249, 109-127.
5. Streptomyces griseus. Retrieved April 6, 2010, from Wikipedia: http://en.wikipedia.org/wiki/Streptomyces_griseus