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Biotechnology summary
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This document contains a brief summary of all the basic principles of the course "biotechnologie".
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Biotechnology 3) Transformation E.coli with heat shock orelectroporation for plasmid copies.
Molecular cloning 4) Transfection in expression vector
Molecular cloning refers to the
isolation of a DNA sequence Molecular cloning with PCR
and insert it into a vector The Gateway cloning technique does not need
without the alteration of the digestion/ligation but uses a bacteriophage to get viral DNA
original sequence (in frame into a bacterial genome by exchanging short ATT sequences
cloning). The vector can be and therefore creating a prophage. An advantage of this
placed in a host cell for copies method is that it is easy to transfer a gene into a variety of
and expression of the gene of vectors.
interest.
Cloning vector characteristics
Genomic libraries o ORI
Complete collection of cloned DNA fragments of an entire o Selectable marker
organism. First, the genome is cut into pieces using o Multiple cloning site/polylinker
restriction endonucleases or shearing (using mechanical
instruments to cleave DNA), all fragments are ligated into Types of cloning vectors
different vectors with ligase. The vector is transferred into o Plasmids
E.coli making a library where the entire genome is spread o Bacteriophages
out over thousands of bacterial colonies. Proteins produced o Bacterial artificial chromosomes (BACs)
by the library can be detected using an immunoassay o Yeast artificial chromosomes (YACs)
(hybridisation, blotting, and detection with probes). o Viral vectors (SV40, adenovirus)
Plasmid as cloning vector
Plasmids are double-stranded circular DNA molecules that
self-replicate. They are small, easy to purify, but only useful
How to make cDNA
mRNA is isolated and cDNA is made using reverse for smaller fragments.
transcriptase using oligo (dT) nucleotides. cDNA can also be
produced naturally by retroviruses and integrated into the Bacteriophage as cloning vector
host’s genome creating a provirus. When a certain cDNA is Engineered version of bacteriophage that infects E.coli. The
needed, the cDNA back is explored with colony phage chromosome is linear, and the DNA is packages in
phage heads from virus particles. They replicate in E.coli
hybridisation to find the gene of interest. The bacteria are
plated and put on a membrane. The bacteria are lysed, and using the lytic cycle.
the cDNA is denaturated to form single strand DNA. A
radioactive probe is added that hybridises with the gene of Expression vector characteristics
interest. These can be used for further isolation and o ORI that allows replication within the cell, so a
prokaryotic cell needs a prokaryotic ORI, and a
amplification.
eukaryotic cell needs an eukaryotic ORI.
Gene isolation o Promoter that can be switches on and off.
Before a gene can be inserted into a vector, it’s isolated by o Selection markers
extracting mRNA and making a cDNA library. Random o Terminator
clones can be isolated from the library and identified with o Polylinker/multiple cloning site.
sequencing. cDNA is then copied using PCR+ specific
primers. Types of expression vectors
o Bacterial expression system
Primer design o Yeast expression system
A primer must consist of a leader sequence, which adds o Viral expression system
extra base pairs on the 5’- end of the primer to assist the o Mammalian expression system
o Transgenic plants
restriction ligation. Also, a restriction site for the enzymes
o Cell-free expression system
and a hybridisation site that binds to the amplificon. The
sequence is always written from 5’- to 3’-, meaning the
primer must be written in that direction too. Choosing an expression system
FW primer: leader sequence – restriction enzyme – DNA of Large proteins Eukaryote
interest + start codon Small proteins Prokaryote
RV primer: the same, but no stop codon and the sequence Glycosylation Baculovirus or mammalian
is written in the reversed direction. 5’ – 3’ High yields, low costs? E.coli, transgenic plants
PTM Yeast, baculovirus, mammalian
Molecular cloning with restriction enzymes
1) Digestion with restriction enzymes
2) Ligation with T4 ligase
, Shuttle vectors Eukaryotic expression systems
These are capable of replicating in two or more types of Yeast expression systems
hosts. They replicate autonomously or integrate into the o S. cerevisiae
host genome and replicate when the host replicates. They o YIP integrated into the genome, double crossing-over.
are used for transporting genes from one organism to o YEP Episomal
another. o YAC Episomal, designed to clone large segments.
Prokaryotic expression systems Yeast expression system
o E.coli expression system Yeast can carry out PTM and secrete eukaryotic proteins.
o T7 expression system They are also easy to manipulate, do not contain toxins,
o pET system grow rapidly, are cheap, have high expression levels, lots of
mutans are known, easy to plate, high copy number, and
Fusion tag are safe in use. Though, the secretion is not optimal,
Protein or peptide fused to the protein of interest for there is codon bias, and the PTM is not always correct
expression measurement, looking at cell location, (incorrect glycosylation and S-S bonds, which causes
purification, signal sequence for sorting or secretion, unstable proteins).
improve solubility, higher stability, targeting for The host chromosome replicates independently via a single
biopharmaceuticals. They can also be used for processing ORI called autonomous replicating sequence (ARS).
by mutating the transcription factor Xbp-1, which improves
folding and secretion. Yeast vector
2pm circle plasmid, extrasomal. These expression plasmids
Constitutive promoter are usually shuttle vectors that can be propagated both in
This is a promoter that is always on. yeast and bacteria. The plasmid itself contains an ORI and 4
genes: REPI and REPII (proteins for plasmid replication),
pET system FLP( protein for intramolecular recombination) and
Advantages: strong expression (50% of total protein in D (function unknown).
culture) and tightly controlled expression (system is off in
absence of IPTG). A disadvantage of this system is that it is Transformation of yeast cells
a leaky expression system, meaning that even in the o Electroporation
absence of IPTG, there may be some low-level o Lithium acetate + heat shock
expression of T7 RNA polymerase from the Lac promoter in o Cell wall removal with B. glucanase or B-glucuronidase
some expression host strains, which could lead to bacterial
toxicity for certain genes of interest in certain host strains. Selection of yeast cells
Auxotrophic selection: auxotrophic genes need a specific
T7 expression system growth substance beyond the minimum required for
The pET expression vector is engineered to integrate the T7 normal metabolism and reproduction by the parental or
promoter + gene of interest. The vector is then transformed WT-strain. The WT strain can make leucine by
into E.coli, which possesses T7 RNA polymerase. The T7 themselves. The leucine-2 mutant can only survive when
gene is under control of the Lac promoter (IPTG). The there is leucine in the environment, meaning that only
system can be used to produce toxic proteins very fast. transformed cells can survive. The auxotrophic mutants can
be screened via replica plating. This is a technique in which
Inducible promoters one or more petri dishes contain selective growth media
Are switched on and off by transcription factors, and are inoculated with the same colonies of
depending on the in vitro conditions. An example is the lac microorganisms from a primary plate, reproducing the
operon that only turns on when lactose is present and original pattern of colonies. It involves a velveteen-covered
other sugars are absent. The inducer is allolactose (IPTG), disk that is pressed onto another plate. The purpose of this
which is a modified form of lactose. Also, Tetracycline is an replica plating is to be able to compare the plates to screen
inducible promoter. for a desired phenotype.
P1-promoter T-vector systems
This is a promoter from the bacteriophage lambda. Used to clone PCR products. Tap polymerase always ends
Expression is repressed when the temperature is around 28 with a 3'-A. The T-rector has a T-overhang, meaning that
degrees, but the expression is induced when the the insert + vector fit perfectly.
temperature exceeds 42 degrees.
Transformation and selection of yeast shuttle vectors
Transformation First, the shuttle vector is transformed into E.coli and
o Electroporation selected with ampicillin to select which bacteria have
o Heat shock (CaCl2) absorbed plasmids. The second selection marker,
To incorporate the antibiotic resistance gene, the DNA is tetracycline is used to see if there is an insert in the
integrated into the genome via double cross-over. plasmid. When you found the construct of interest it can be
amplified and brought into the host cell. The host cell is
then selected with a metabolic selection marker to select
the cells that have taken up the construct.
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