Biotechnology of drugs – Basics of genetic
engineering I.
Doc. RNDr. Jan Hošek, Ph.D.
hosekj@pharm.muni.cz
Department of Molecular Pharmacy
FaF MU
Interesting site
http://learn.genetics.utah.edu/
https://learn.genetics.utah.edu/
1. By classical biotechnological procedure
▪ interbreed
▪ mutagenesis of a population of cells
▪ selection of cells/organisms with suitable properties
▪ they cannot force the organism to produce a protein that is not its own
2. By genetic engineering - recombinant DNA technology
▪ gene cloning → recombinant DNA technology
▪ genetic manipulation in vitro
▪ breaks down barriers between species → heterologous systems
Biotechnological product can be created:
The effort to genetically encode the synthesis of a commercially
advantageous product into an organism that will produce it cheaply
and with high yield.
The goal of recombinant technologies
2000 kg of pancreas
200 mL of insulin
Recombinant technology - gene cloning - needs the incorporation
of foreign DNA into the host cell
➢ introduction of DNA (transformation) into the cell
➢ ensuring the survival of forein DNA
➢ ability to replicate
➢ assurance of expression
Basic principles of recombinant technologies
➢ own gene – DNA fragment
➢ vector – plasmid, phage,
cosmid
➢ host – recipient of
recombinant DNA
➢ insert = gene incorporated into
the vector
➢ recombinant DNA = vector with
insert
What is needed for gene cloning
https://microbenotes.com/gene-cloning-requirements-principle-steps-applications/
A permanent heritable change in the genetic material of a cell caused
by the acceptance and incorporation of foreign genetic information
Sources of foreign genetic information
animal
vegetal
microbial
synthetic
➢ ability to replicate
➢ ability to express
Consequence of gene cloning = transformation
➢The recombinant protein can be:
▪ Final product (e.g. hormone, antibody)
▪ Tool for further synthesis (e.g. various enzymes)
How to get a gene or its fragment?
Isolate DNA in its native state
(see Methods of mol. biol.)
Process enzymes Perform PCR
Direct synthesis
Clone only
And how to proceed further?
2) Do I have it in sufficient quantity and quality?
a) Process enzymes (see Methods of mol. biol.)
b) Perform PCR (see Methods of mol. biol.)
YES
NO
1) Did I get a nucleic acid?
YES
NO
Factors influencing the expression of cloned genes
Regulatory sequences for gene
expression
1. Transcription
a. The power of the promoter
b. Transcription terminator
c. Stability of mRNA
2. Translation
a. Structure of the ribosome binding site
b. Codon usage
3. Transport of proteins
a. Character of the signal sequence
Properties of vectors
1. The number of copies of the vector
in the cell
2. Vector stability
Physiology of the host cell
1. Growing conditions
2. The enzyme apparatus of the host
cell
Insert properties
• It contains a coding
sequence
• Contains the start of
translation (ATG start
codon)
• Contains the end of
translation (stop codon)
• It carries the
appropriate codons for
specific amino acids
Signals influencing prokaryotic structural gene
transcription and translation
regulatory signals
for transcription
regulatory signals for
translation initiation Cloned gene
Leading sequence
Signal peptide –
transport
regulatory
sequence of
the vector
Properties of vectors
Cloned structural gene, cDNA
Power of promoter
Terminator of
transcription
1. signals for translation initiation
2. signals for transport
Codon usage
Leading
sequence
1. secondary mRNA structure
2. signals for splicing (3‘ and 5‘ end of intron)
Promoter
The effect of the distance between the promoter and the start
of translation on the secondary structure of mRNA and on
the amount of expressed protein
Deletions of
varying
extent
Relative amount of CRO protein
produced
Distance variability Secondary structure of cro-mRNA
Relative amount of
CRO protein produced
Possibilities of ensuring high proteosynthesis
• The rate of proteosynthesis depends on
the amount of mRNA in the cell
• Modifications in the 3'-UTR or 5'-UTR can
increase the mRNA half-life
• The PuPuUUUPuPu sequence near the
Shine-Dalgarn sequence is essential for
translation of eukaryotic genes in E. coli
• Construction of homopolycistronic
expression cassettes
• Proteins in the periplasm have a longer
half-life than in the cytoplasm
• Targeted inhibition of specific proteases
• Formation of fusion proteins – protein
from the host organism (stabilizing
partner) + protein of interest
Synthetic ribosome binding site
(RBS)
S-D sequence
Stop codone TAA
Cloned gene
coding sequencepromoter
PstI
recognise site
HindIII recognise site
1 promoter
1 terminator
Many copies of GOI
Effect of codon usage
• Different species use
codons with different
frequencies → related to
the amount of tRNA
• Solution:
– Cloning rare tRNAs
together with a gene of
interest
– Substitution of rare
codons for common ones
by site-directed
mutagenesis
http://2014.igem.org/Team:Penn_State/CodonOptimization
➢ Plasmids
➢ Bacteriophages
➢ Cosmids
➢ Artificial chromosomes
➢ BAC (bacterial artificial chromosomes)
➢ YAC (yeast artificial chromosomes)
Vectors
https://www.sciencedirect.com/topics/nursing-and-health-
professions/artificial-chromosome
Anatomy of an expression vector
1) origin of replication (ori), which is a condition for the production of new
copies
2) inducible promoter, which will allow to regulate the expression of the desired
protein
3) selection marker ensuring the preferential growth of transformed bacteria
4) multi-cloning site (MSC), allowing a foreign DNA fragment to be inserted into
the plasmid
Front. Microbiol., 17 April 2014 |
https://doi.org/10.3389/fmicb.2014.00172
Examples of regulatable promoters in
expression vectors
Promoter Origin Way of regulation
independence
from catabolic
repression
utilization in
eukaryotes
➢ extrachromosomal circular
dsDNA
➢ occurrence in many bacterial
species
➢ 1,000 to 200,000 bp in size
➢ carry only genes encoding
secondary traits (resistance
to antibiotics)
➢ autonomous replication
➢ insert size = up to 25 kbp
Plasmids as vectors
Recent trend →
plasmid synthes on demand
1) small size = ability to transform
2) plasmid stability
3) high number of copies in the cell = yield
4) easy handling
5) "shuttle" vectors = work in multiple host
species (e.g. E. coli + mammalian cells)
Plasmid suitability criteria
https://www.genome.gov/genetics-glossary/Plasmid
bla (ApR)
rep
MCS
lacZα
Plasmids pUC18 and pUC19
MCS = multi-cloning site
Polylinker pUC18 and pUC19
Expression plasmid pIRES2-eGFP
Bacteriophage λ-based vectors
➢ hey replace plasmids when
longer DNA fragments need to
be cloned
➢ Insertion vectors 8 – 10 kbp
➢ Replacement vectors 8 – 24
kbp
https://bio3400.nicerweb.com/Locked/media/ch19/lambda-vector.html
➢ 50 kbp dsDNA
➢ linear and circular
form
➢ cos sites
Bakteriophage λ
http://hotcore.info/babki/bacteriophage-lysogenic-cycle.html
https://doi.org/10.1016/C2009-0-01986-2
Cosmids
➢ combination of plasmid and
phage
➢ prokaryotic origin of replication
oriV
➢ selection marker
➢ cloning site
➢ capacity 37 - 52 kbp
➢ cos sites of bacteriophage λ
➢ for packaging, packaging
proteins must be added
➢ enters as a phage
➢ it behaves like a plasmid in the cell
➢ BAC = bacterial artificial chromosome
➢ Derived from plasmid F'
➢ Designed for cloning into bacterial cells
➢ They occur in the number of 1-2 copies
per cell
➢ Cloned DNA is highly stable
➢ Cloning capacity up to 300 kbp
(maybe more)
➢ Used in the HUGO project
➢ Today, they are replaced by the methods of
whole-genome sequencing, next generation
sequencing and third generation sequencing
Bacterial Artificial Chromosome
https://library.uams.edu/assets/COM/BioChem/MolecularTools/
MolecularToolsSDL12.html
Phagemid
➢ Plasmid containing origin of replication of phage M13
➢ It is used to prepare ssDNA.
➢ The best known examples are the pBluescript series of cloning vectors.
Other variants of vectors
Phosmid
➢ Similar to a cosmid, but based on a bacterial F-plasmid
➢ The host (E. coli) may contain only a single molecule
➢ Cloning capacity up to 40 kbp
➢ Suitable for constructing stable libraries from complex genomes
➢ Highly stable; capable of maintaining human DNA for over 100 generations
(A) Typical bacterial cloning vector. This vector has bacterial
sequences to initiate replication and transcription. In addition, it has
a multiple cloning site embedded within the lacZ α gene so that the
insert can be identified by alpha-complementation. The antibiotic
resistance gene allows the researcher to identify any E. coli cells that
have the plasmid. (B) Yeast shuttle vector. This vector can survive in
either bacteria or yeast because it has both yeast and bacterial
origin of replication, a yeast centromere, and selectable markers for
yeast and bacteria. As with most cloning vectors, there is a
polylinker. (C) Lambda replacement vectors. Because lambda phage
is easy to grow and manipulate, its genome has been modified to
accept foreign DNA inserts. The region of the genome shown in
green is nonessential for lambda growth and packaging. This region
can be replaced with large inserts of foreign DNA (up to about 23
kb). (D) Cosmids. Cosmids are small multicopy plasmids that
carry cos sites. They are linearized and cut so that each half has
a cos site (not shown). Next, foreign DNA is inserted to relink the two
halves of the cosmid DNA. This construct is packaged into lambda
virus heads and used to infect E. coli. (E) Artificial chromosomes.
Yeast artificial chromosomes have two forms: a circular form for
growing in bacteria and a linear form for growing in yeast. The
circular form is maintained like any other plasmid in bacteria, but the
linear form must have telomere sequences to be maintained in
yeast. The linear form can hold up to 2000 kb of cloned DNA and is
very useful for genomics research.
https://doi.org/10.1016/C2009-0-64257-4
Regardless of source type
➢ bacterial cells
➢ yeasts and molds
➢ plant and animal cells
➢ whole plant or animal
Hosts = recipients of recombinant DNA
➢ G- bacteria, circular chromosome 3×106 bp
➢ amount of usable plasmids
➢ generation time 20 min. → rapid biomass
formation
➢ undemanding and cheap cultivation
➢ stationary phase 2×109 cells/mL
➢ a number of mutants (DH5α, HB101,
BL21,…)
➢ disadvantages – significantly different posttranslational
modifications compared to
eukaryotes
Escherichia coli
Saccharomyces cerevisiae
➢ linear chromosomes
➢ approximately 13×106 bp
➢ about 6,275 genes
➢ Schizosaccharomyces pombe
➢ Pichia pastoris
➢ the simplest eukaryotic organism
➢ identical transcription and translation
apparatus with other eukaryotes
➢ differences in post-translational processes,
e.g. mannose hyperglycosylation
➢ similar selection principles as in lower eukaryotes or bacteria
➢ use of binary "shuttle" vectors (bacteria + insects)
➢ replacement of the polyhedrin sequence of the virus with a recombinant
gene
➢ the most commonly used Sf9 cell culture derived from Spodoptera
frugiperda
➢ baculoviruses only attack insect cells
Insect cells and baculoviruses
https://www.genscript.com/insect-customized-expression-package.html
➢ vectors are typically bacterial plasmids that contain plant
expression cassettes
Relatively safe technology
➢ direct transformation
➢ transformation using Agrobacterium
➢ transformation by viral vector
➢ host cells – Nicotiana tabacum,
Arabidopsis thaliana, …
Plant cells
➢ systems closest to man
➢ the most common producer is mammalian CHO cells (Chinesse
hamster ovaries)
➢ differential interspecies glycosylation
Technology security issues !
➢ as vectors serve adenoviruses, retroviruses,
herpesviruses
Mammalian cells and their viruses
Creation of recombinant proteins in
different organisms