1. komponenta

Lecture type	Total
Practicum	60

* Load is given in academic hour (1 academic hour = 45 minutes)

COURSE CONTENT:
PCR amplification of the DNA fragments coding the gene for the protein of interest. Plasmid DNA isolation and restriction digestion. Ligation of the amplified fragment into the prepared plasmid vector. Selection of the recombinant plasmid. Transformation of the bacterial strain with the recombinant plasmid. Induction of the overexpression of the protein coded by the amplified DNA fragment. Analysis of the protein overexpression by SDS gel-electrophoresis. Isolation of the total cellular proteins from the induced bacteria. Purification of the overexpressed protein by affinity chromatography on the Ni-NTA agarose column. Analysis of the chromatographic fractions on the SDS polyacrylamide gel. Western analysis. Exchange of the buffers using dialysis and gel-filtration chromatography. Concentration of the purified protein. Spectrophotometric determination of the protein concentration.

LEARNING OUTCOMES:
1. to independently perform complex and modern biochemical and molecular-biological experiments that include techniques of recombinant DNA and methods of protein manipulation
2. to independently use modern equipment in biochemical laboratory
3. to design procedure for gene cloning into plasmid vector in order to obtain recombinant fusion protein
4. to perform gene cloning experiments including the use of appropriate experimental controls
5. to transform bacterial cells with recombinant plasmid
6. to overexpress recombinant fusion protein in bacterial host
7. to purify overexpressed protein using affinity chromatography
8. to determine purity and concentration of purified protein
9. to critically evaluate performed experiments and to recommend/suggest improvements and/or optimization of experimental conditions and procedures
10. to write a detailed report in the form of scientific paper

1. OBAVEZNA LITERATURA:
   Interna skripta za Viši praktikum biokemije (2. radno izd.), Zavod za biokemiju, PMF, Zagreb 2013.
   pET System Manual (11. izd.), Novagen, 2005.
   Relevantna poglavlja iz knjige R. R. Burgess, M. P. Deutscher (ur.), Methods in Enzymology: Guide to Protein Purification (2. izd.), Elsevier, 2009.
   Odabrani izvorni i revijalni znanstveni radovi
2. DOPUNSKA LITERATURA:
   A. Ambriović Ristov (gl. ur.), Metode u molekularnoj biologiji, Institut Ruđer Bošković, Zagreb, 2007.
   J. Sambrook i D. W. Russell, Molecular Cloning: a laboratory manual (3. izd.) Cold Spring Harbor Laboratory Press, 2001.
   S. B. Primrose i R. M. Twyman, Principles of Gene Manipulation and Genomics (7. izd.), Blackwell Publishing, 2006.
   L. Stryer, J. Berg i J. Tymoczko, Biokemija (6. izd), Školska knjiga, 2013. (prijevod na hrvatski jezik)
   J. M. Berg, J. L. Tymoczko i L. Stryer, Biochemistry (7. izd.), W. H. Freeman & Co, New York, 2012.
   D. L. Nelson i M. M. Cox, Lehninger Principles Of Biochemistry (6. izd.), W. H. Freeman & Co, New York, 2013.

Izborni predmeti izvan odabranih grana - Regular study - Analytical and inorganic chemistry
Izborni predmeti izvan odabranih grana - Regular study - Analytical and organic chemistry
Izborni predmeti izvan odabranih grana - Regular study - Analytical and physical chemistry
Mandatory course - Regular study - Analytical chemistry and biochemistry
Mandatory course - Regular study - Biochemistry and organic chemistry
Mandatory course - Regular study - Biochemistry and physical chemistry
Izborni predmeti izvan odabranih grana - Regular study - Inorganic and organic chemistry
Izborni predmeti izvan odabranih grana - Regular study - Inorganic and physical chemistry
Mandatory course - Regular study - Inorganic chemistry and biochemistry
Izborni predmeti izvan odabranih grana - Regular study - Physical and organic chemistry

Izborni predmeti izvan odabranih grana - Regular study - Analytical and inorganic chemistry
Izborni predmeti izvan odabranih grana - Regular study - Analytical and organic chemistry
Izborni predmeti izvan odabranih grana - Regular study - Analytical and physical chemistry
Izborni predmeti izvan odabranih grana - Regular study - Inorganic and organic chemistry
Izborni predmeti izvan odabranih grana - Regular study - Inorganic and physical chemistry
Izborni predmeti izvan odabranih grana - Regular study - Physical and organic chemistry