MICROBIAL PHYSIOLOGY IN THE GENOMIC ERA:
A REVOLUTIONARY TALE
PART 2
Roga F. Kembaren, M.Si
Dosen Teknik Bioproses
TRADITIONAL TOOLS
Mutant Hunts
Reporter Genes
Polymerase Chain Reaction (PCR)
DNA Mobility Shifts
GENOMIC AND
PROTEOMIC TOOLS
Southern Blot
Northern Blot
Western Blot
Southwestern Blot
Two-hybrid System
1. Understanding the principle of genomic and
proteomic tools.
2. Able to distinguish the principles and purposes of
each technique used for genomic and proteomic.
OUTCOMES
3. Able to determine the right technique for genome
and proteome studies related to microbial
physiology.
• The classical approach used to expose the details of biochemical
pathway requires the presence of selectable phenotype
- such as an ability to grow on a carbohydrate
• Mutants that have lost this phenotype are then sought.
• The biochemical step in the pathway is explored and encoding gene
can be mapped, identified, cloned and sequenced.
MUTANT HUNTS
Replica plating
https://www.magazinescience.com/en/biology/isolation-of-mutant-bacteria/?print=print)
• Flow cytometry enables the simultaneous detection of
multiple surface and intracellular antigens for proteomic
profiling of cells.
• This allows characterization and identification of specific
cell subtypes within a heterogeneous population and is
usually called immunophenotyping.
FLOW CYTOMETRY • Antigen-specific antibodies, conjugated to various
fluorophores, are incubated with the sample to identify
each marker.
• Fluorescent light of various wavelengths can be
separated, detected, and converted into a digital signal in
a flow cytometer.
Methods Mol Biol 2021;2261:213-227.
doi: 10.1007/978-1-0716-1186-9_13
Fluorescent-activated cell sorting (FACS) - Flow cytometer
Separates cells that are labeled
differentially with a fluorescent reagent.
1. Concentrated suspension of labeled
cells is mixed with buffer (sheath fluid).
2. Both fluorescent light emitted and the
light scattered by each cell are
measured.
From measurement of the scattered light,
the size and shape of the cell can be
determined.
3. Given a negative electric charge
proportional to the amount of
fluorescence of its cells.
4. Separation of no charge cells and cells
with different electric charges.
Adapted from D.R. Parks and L.A. Herzenberg, 1982, Meth. Cell.Biol. 26:283
Transcriptional and Translational Gene Fusions.
(Reporter genes)
Used to analyze various aspects of gene expression
Reporter Genes
Examples of reporter gene:
- lacZ (gene encoding -galactosidase)
- gfp (gene encoding green fluorescent protein)
Reporter Genes
Moat et al. 2002. Microbial Physiology 4ed.
• To amplify a specific-size DNA
• Can be used to isolate specific
DNA in a large amount.
POLYMERASE
CHAIN REACTION • Can be used to detect the
(PCR) presence of that organism in a
complex environment.
Moat et al. 2002. Microbial Physiology 4ed.
PCR-mediated site-directed mutagenesis
• PCR can also be used to
engineer specific mutations
in a given gene (site-directed
mutagenesis).
• Using mutation primers
PCR
Moat et al. 2002. Microbial Physiology 4ed.
• To identify whether the putative
(or predicted) regulatory protein
directly bind to the target DNA
sequence.
• Using agarose electrophoresis or
polyacrylamide electrophoresis.
DNA Mobility Shifts
(Gel Shifts and Supershifts)
Electrophoretic mobility shift and supershift.
Moat et al. 2002. Microbial Physiology 4ed.
Finding Transcriptional Start by Primer Extension
Genomic Tools
Southern / Northern Blot
To detect specific DNA / RNA
Gerald Karp (2013) Cell and Molecular Biology: Concepts and Experiments 7th ed.
• To detect specific protein in a
mixture.
• Protein extract is subjected to
SDS-PAGE.
• Separated proteins on the gel
are transferred into PVDF
membrane.
Western Blot • Membrane is probed with
primary antibody.
• Protein that bind specifically to
the primary antibody is
visualized with the labeled
secondary antibody.
Moat et al. 2002. Microbial Physiology 4ed.
Southwestern blot
• Can be used to identify proteins that bind specific DNA.
• Proteins are separated using SDS-PAGE.
Southwestern blot • Separated protein bands are transferred to a nitrocellulose
membrane.
• Membrane is probed with labeled oligonucleotide.
• Identify the specific protein band that binds the labeled
DNA. .
• The two-hybrid system is an artificially constructed
genetic system intended to facilitate the detection and
assessment of protein-protein interactions
• In the two-hybrid system a host organism, typically
yeast or bacteria, is engineered so as to contain three
components.
(1) protein fused to a DNA-binding domain of known
Two-hybrid system specificity (hybrid 1);
(2) protein fused to a transcriptional-activation domain
(hybrid 2), that can interact with the first protein,
constituting a functional, albeit composite, transcription
factor;
(3) one or more reporter genes transcribed based on the
binding of the composite transcription factor.
Serebriiskii, ... E.A. Golemis, in Encyclopedia of Biological Chemistry
(Second Edition), 2013
Yeast two-hybrid analysis
• The goal is to bring together the
DNA binding domain and the
activation domain of the yeast
Gal4 protein through the
interaction of proteins X and Y, to
Two-hybrid system which one or other of the domain
is fused.
• The interaction is accompanied
by the expression of a reporter
gene.
Lehninger (2017) Principles of Biochemistry, 7th ed.
Two-hybrid system
• Bacterial two-hybrid system involving Bordetella pertussis adenylate
cyclase.
• Interaction between X and Y fusion proteins brings together the T18
and T25 moieties of B. pertussis adenylate cyclase.
• The resulting production of cAMP can be measured through
expression of a cAMP-dependent gene (e.g. lacZ)
Moat et al. 2002. Microbial Physiology 4ed.
REFERENCES
1. David L. Nelson and Michael M. Cox, 2017, Lehninger: Principles of
Biochemistry, 7th ed., Freeman Company, New York, USA.
2. Gerald Karp (2013) Cell and Molecular Biology: Concepts and
Experiments 7th ed. John Wiley and Sons, USA
3. Lodish H. et.al. (2008) Molecular Cell Biology 5th ed. W.H.Freeman
and Company, USA.
4. Moat et al. 2002. Microbial Physiology 4ed. Jihn Wiley and Sons, USA.
