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Summary- Applied molecular microbiology (MIB30806)
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Summary- Applied molecular microbiology (MIB30806) This summary covers the entire reader of the course, including a descriptive CRISPR-Cas explanation.
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Applied molecular microbiologysummary
Chapter 1
Types of biotechnology:
Green: agro-food
Red: medical
Blue: marine
White: general industrial applications
Nanotechnology: integration of physics and chemistry, to control matter on the atomic and molecular
scale. Bio-nanotechnology: when biomolecules are involved.
The stages of biotechnology development:
1. Classical fermentation (before 1865): alcoholic beverages and dairy products
2. Fermentation products (1865-1940): solvent fermentation and organic acid fermentation
3. Antibiotics (1940-1960): large-scale production, microbial steroid transformations
4. Amino acids and enzymes (1960-1975): production of industrial enzymes and amino acids
5. Genetic engineering (1975-1995): recombinant DNA technology
6. Genomics and synthetic biology (1995): first microbial genome
Louis Pasteur (in 1863): proofed that living microbes are the active agents of fermentation.
Recombinant DNA technology: can be used to introduce entirely new synthetic capabilities in cells.
Primary metabolites: fermentation products. Secondary metabolites: antibiotics.
Chapter 2
DNA: the carrier of genetic material in all prokaryotic and eukaryotic organisms. DNA is a linear
polymer with repeating units of nucleotides, consisting of a sugar-part (deoxyribose), and a variable
base-part (A, C, T or G). The DNA strands form a double helix.
Phosphate-group: links between the 3’ and the 5’ carbon atom of two deoxyriboses. The sugar-
phosphate backbone is often outside of the double helix.
A requirement for base-pairing is that both strands are anti-parallel and complementary.
RNA: has a similar structure, however deoxyribose is replaced by ribose and T is replaced by U.
Central dogma: DNA is transcribed to mRNA, which is translated to proteins by rRNAs.
rRNA: the catalytic component responsible for peptide bond synthesis in the frame of a ribosome.
tRNA: specific in terms of mRNA codon recognition and amino acid delivery.
DNA polymerase: catalyses the addition of deoxyribonucleotide triphosphate (dNTPs) during DNA
synthesis. Single stranded DNA is used as a template and synthesis often starts with an
oligonucleotide primer (with a free 3’ hydroxyl group).
Oligonucleotide synthesis: addition of dNTP monomers to a growing oligonucleotide chain, linked
with the 3’-end to a solid phase support. The 3’-phosphorus atom of the incoming monomer is linked
to the 5’-oxygen atom of the growing chain.
PCR: high temperatures are used to melt the double stranded DNA, so heat-stable DNA polymerases
are also used (Taq polymerase). The generated DNA strand becomes the template for replication in
the next cycle.
DNA sequence analysis: DNA sequencing is the process of determining the nucleotide order of a
, certain DNA fragment. Sanger method:
uses sequence specific termination of a
synthesis reaction using ddNTPs as
substrates, lacking the 3’-hydroxyl
group. When incorporated, they
terminate synthesis since no more
nucleotides can be attached. The result
is a mixture of fragments of different
sizes. ddNTPs can also be labelled with
fluorescent dyes, where the order of
colours can determine the sequence.
Pyrosequencing: a sequencing technique
based on the detection of released
pyrophosphate (PPi) during DNA
synthesis. The release of PPi results in
the generation of visible light that is proportional to the number of
incorporated nucleotides. This is because PPi is converted to ATP by
ATP sulfurylase, which provides the energy to luciferase to oxidize
luciferin and generate light. Pyrosequencing can be performed in a
solid-phase or a liquid-phase. The liquid-phase sequencing method
adds another enzyme (apyrase) so that the reaction does not need a
washing step and can be performed in a single tube.
RNA synthesis: the activated monomer substrates are ribonucleotide
triphosphates (NTPs). RNA polymerases catalyse the synthesis and don’t need primers to do so. RNA
polymerase has no proofreading activity. Recognition of transcription start sites is generally
controlled via specific protein-DNA (promotor) interactions.
Archaea and eukaryotes: the docking of the TATA-binding protein to the TATA-box.
Bacteria: recognition of the region by the sigma subunit of RNA polymerase.
Protein synthesis/translation: mediated by the interplay of mRNA, rRNA, tRNA, aminoacyl-tRNA
synthetases, and protein factors. Protein synthesis takes place on a ribosome consisting of a small and
large subunit. The start signal on mRNA is usually AUG, preceded by a purine-rich sequence (GGTGA
or ribosome binding site, RBS). During translation, the codons on the mRNA interact with the
anticodons of the tRNA. Some tRNAs can interact with more than one codon, since pairing of the
third base is less crucial than the other two (wobble mechanism).
Codon: a triplet of nucleotides encoding for an amino acid. There are 64 amino acids, of which 3 are
stop codons.
Synonymous codons: all amino acids (except methionine and tryptophan) are encoded by more than
one codon.
Codon bias/codon usage bias: an uneven distribution of codon use for each of the 18 amino acids. A
tRNA corresponding to a rarely used codon generally occurs in relatively low intracellular
concentrations. Optimal codons are well-recognised by tRNA and result in fast translation. The
opposite is true for non-optimal codons. The codon bias differs per organism.
Loop regions: connecting secondary structure elements such as alpha-helices and beta-strands. A lot
of non-optimal/slow codons are present in this region, contributing to co-translational folding of
these protein domains. One or more non-optimal codons at certain regions in the mRNA sequence
are needed to slow down the rate of elongation. The optimal position of fast and slow codons can not
easily be predicted.
, Chapter 4
Benefits of using E. coli as an expression system:
Availability
Feasibility: the standard recombinant DNA techniques necessary to express reasonable
protein amounts are relatively simple
Efficiency: high yields, fast, and cheap
Disadvantages:
No capacity for post-translational modifications
Proteins produced in large amounts often form aggregates, inclusion bodies
It is difficult to arrange the secretion of large amounts of expressed proteins
Plasmids: self-replicating, extra chromosomal DNA molecules. All plasmid vectors contain a replicator,
a selectable marker, and a cloning site.
Replicator: a stretch of DNA that contains the site at which DNA replication begins (the origin of
replication), and that may include genes encoding specific replication factors.
Copy number: refers to the number of plasmid molecules maintained per bacterial cell. High-copy
number: more than 15 plasmids. Low-copy number: used when you need to control the gene dosage
of a cloned sequence (toxic).
Relaxed control: for high-copy numbers. The plasmids initiate replication in a process controlled by
plasmid-encoded functions. Stringent control: for low-copy numbers. Initiation of replication depends
on unstable proteins synthesized at the start of the bacterial cell cycle.
Toxin/antitoxin system (ccdB/ccdA): allows post-segregationally
killing of plasmid-free daughter cells (=suicide vector, see image).
Selectable marker: necessary for following and maintaining the
presence of the plasmid in the cell. The marker is usually dominant
and antibiotic resistant (ampicillin, tetracycline, kanamycin, and
chloramphenicol). Sometimes, recessive markers are used. A leuB-
deficient marker can for example not grow in the absence of
leucine.
Cloning site: restriction endonuclease cleaving site into which
foreign DNA can be inserted. Cutting the vector with a restriction
endonuclease does not disrupt other critical features of the vector
(unique). The sequences that directly flank the polylinker (multiple
cloning site) are often used for analysis or DNA manipulation (PCR).
What to consider when cloning? The size of the vector, the copy number, the polylinker, and the
ability to select/screen for inserts. Large plasmids give lower DNA yields.
Additional vector features:
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