Molecular biology COURSE 8BM
Lesson 1: introduction into biotechnology
A breakthrough was the production of chymosin which is a protease. It is a key component of rennet.
Rennet is a complex set of enzymes in the stomach. It is used to separate milk and make cheese.
Chymosin curdles casein in milk.
Ancient fermentation: yeast is one of the oldest micro-organisms that have been exploited by
humans. They combined it with fruit, resulting in wine or beer
Classical fermentation: basics for the transfer of genetic information were revealed. From 1800-
1950. During this period pasteurisation was discovered. Furthermore it was demonstrated that
fermentation was a result of the action of living micro-organism.
Glucose is the main carbon source for the micro-organisms. This will be converted to pyruvate. Than
after you add this to different micro-organisms you get different end results like lactic acid, ethanol
CO2 and carbon dioxide .
Modern biotechnology: discoveries have unlimited implications and applications genetic
engineering.
Drosophila and zebrafish are useful model organisms.
With genetic engineering they made the zebrafish see-through so tumor growth can be followed, by
adding GFP you can follow your protein of interest.
Where are we now with biotechnology>?
- Currently available: PCR, CRISPR/Cas9,
PCR (polymerase chain reaction): to make DNA
CRISPR/Cas9: by modifying genomes based on bacterial viral defence system like NHEJ (non-
homologues end joining knock out) and HDR (homology-directed repair knock in)
Bioplastics:
Pretreatment and hydrolosis microbial fermentation separation and polymerization
RED biotechnology: medical
Antibody production: only mammalian cells due to post
translational modifications
To make vaccines: take the antigen producing gene from (for
example) virus. Put it in the plasmid recombinant DNA. Put that
in the recombinant yeast cell. Then it starts producing this antigen
so it can be used as a vaccine.
GREEN biotechnology: agricultural
For example putting an endotoxin in the corn, certain insects can die of this when consumed. No
pests anymore.
You can also do antibody production in plants (for example soy bean seeds). The same concept as in
mammalian cells. The antibodies will stay in the plants. When fed to pigs it can help these pigs to be
resistant to certain things.
,Lesson 2: CRISPR-Cas
We have to make a construct with crispr-cas in the oil yeast
Gene editing, you need cas9 protein, guide RNA and a PAMP site
You have to put it in a vector and do a transformation in the expression host OR into any cell
(does not have to be expression host, can also be the human body: you can inject it). This
makes it different from the general cloning tools
Origin of CRISPR/Cas
Prokaryotic defense mechanisms against plasmids and viruses
Restriction enzymes/nucleases
Acquired immunity: CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas9 system originally discovered in Streptococcus Pyogenes
CRISPR: the immune system of prokaryotes
They found an ‘error’ in the sequence,
repeats. It looks like a virus. Why would a
virus repeat be in a genome? We always
thought that prokaryotes had no immune
system but they do: kill viruses and
Bacteriophage injecting RNA into the
prokaryote.
Specific nucleases: restriction enzymes
If you have your genome and you cut it with
EcoRI? A lot of different cuts
Other site specific nucleases:
Zincfinger ZN and TALENs;
TAL effector nuclease
They recognize a larger
sequence
This is the CAS complex, the bubble is
Cas9 protein. A piece of around 20 bp is
recognized by the genomic DNA of the
cell. Next to that it needs to recognize
another sequence: PAM site.
, -When you compare the tracrRNA and gRNA they are different
pieces, but in the engineered crisprcas it is one piece.
-In the natural crispr you have the bacterial chromosome, in
the engineered version you have a specific plasmid.
The only way cas gets active is when the guide RNA is in place
DNA repair in eukaryotes: non
homologues end joining and
homology directed repair
NHEJ: When the host is
repairing DNA after the cut it
sometimes makes a mutation.
There will be an indel mutation.
This is random, you have no
idea what is going to happen.
You know where: you know
where the cut is. The idea is
that there is a premature stop
codon
When there is a cut at the
beginning: higher chance of
premature stop codon and
most likely to be non-functional
because it’s so short.
HDR: when you add template
DNA, so whilst the host is
repairing the cut, it will take up the template DNA so now you determine the change. You know what
is going to happen when compared to the NHEJ.
So NHEJ is more random and HDR is more specific: host copies the added DNA (precise gene editing)
What do you need? Cas9 protein, sgRNA
(gRNA), target region (20-22 nt), PAM-site,
Target DNA
PAM-site: NGG on sense strand of target DNA
Protospacer Adjacent Motif
PAM site is not in the gRNA, it is a little
docking station in the Cas9 protein. It fins a
lot of NGG
options in the
genome.
Lesson 1: introduction into biotechnology
A breakthrough was the production of chymosin which is a protease. It is a key component of rennet.
Rennet is a complex set of enzymes in the stomach. It is used to separate milk and make cheese.
Chymosin curdles casein in milk.
Ancient fermentation: yeast is one of the oldest micro-organisms that have been exploited by
humans. They combined it with fruit, resulting in wine or beer
Classical fermentation: basics for the transfer of genetic information were revealed. From 1800-
1950. During this period pasteurisation was discovered. Furthermore it was demonstrated that
fermentation was a result of the action of living micro-organism.
Glucose is the main carbon source for the micro-organisms. This will be converted to pyruvate. Than
after you add this to different micro-organisms you get different end results like lactic acid, ethanol
CO2 and carbon dioxide .
Modern biotechnology: discoveries have unlimited implications and applications genetic
engineering.
Drosophila and zebrafish are useful model organisms.
With genetic engineering they made the zebrafish see-through so tumor growth can be followed, by
adding GFP you can follow your protein of interest.
Where are we now with biotechnology>?
- Currently available: PCR, CRISPR/Cas9,
PCR (polymerase chain reaction): to make DNA
CRISPR/Cas9: by modifying genomes based on bacterial viral defence system like NHEJ (non-
homologues end joining knock out) and HDR (homology-directed repair knock in)
Bioplastics:
Pretreatment and hydrolosis microbial fermentation separation and polymerization
RED biotechnology: medical
Antibody production: only mammalian cells due to post
translational modifications
To make vaccines: take the antigen producing gene from (for
example) virus. Put it in the plasmid recombinant DNA. Put that
in the recombinant yeast cell. Then it starts producing this antigen
so it can be used as a vaccine.
GREEN biotechnology: agricultural
For example putting an endotoxin in the corn, certain insects can die of this when consumed. No
pests anymore.
You can also do antibody production in plants (for example soy bean seeds). The same concept as in
mammalian cells. The antibodies will stay in the plants. When fed to pigs it can help these pigs to be
resistant to certain things.
,Lesson 2: CRISPR-Cas
We have to make a construct with crispr-cas in the oil yeast
Gene editing, you need cas9 protein, guide RNA and a PAMP site
You have to put it in a vector and do a transformation in the expression host OR into any cell
(does not have to be expression host, can also be the human body: you can inject it). This
makes it different from the general cloning tools
Origin of CRISPR/Cas
Prokaryotic defense mechanisms against plasmids and viruses
Restriction enzymes/nucleases
Acquired immunity: CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas9 system originally discovered in Streptococcus Pyogenes
CRISPR: the immune system of prokaryotes
They found an ‘error’ in the sequence,
repeats. It looks like a virus. Why would a
virus repeat be in a genome? We always
thought that prokaryotes had no immune
system but they do: kill viruses and
Bacteriophage injecting RNA into the
prokaryote.
Specific nucleases: restriction enzymes
If you have your genome and you cut it with
EcoRI? A lot of different cuts
Other site specific nucleases:
Zincfinger ZN and TALENs;
TAL effector nuclease
They recognize a larger
sequence
This is the CAS complex, the bubble is
Cas9 protein. A piece of around 20 bp is
recognized by the genomic DNA of the
cell. Next to that it needs to recognize
another sequence: PAM site.
, -When you compare the tracrRNA and gRNA they are different
pieces, but in the engineered crisprcas it is one piece.
-In the natural crispr you have the bacterial chromosome, in
the engineered version you have a specific plasmid.
The only way cas gets active is when the guide RNA is in place
DNA repair in eukaryotes: non
homologues end joining and
homology directed repair
NHEJ: When the host is
repairing DNA after the cut it
sometimes makes a mutation.
There will be an indel mutation.
This is random, you have no
idea what is going to happen.
You know where: you know
where the cut is. The idea is
that there is a premature stop
codon
When there is a cut at the
beginning: higher chance of
premature stop codon and
most likely to be non-functional
because it’s so short.
HDR: when you add template
DNA, so whilst the host is
repairing the cut, it will take up the template DNA so now you determine the change. You know what
is going to happen when compared to the NHEJ.
So NHEJ is more random and HDR is more specific: host copies the added DNA (precise gene editing)
What do you need? Cas9 protein, sgRNA
(gRNA), target region (20-22 nt), PAM-site,
Target DNA
PAM-site: NGG on sense strand of target DNA
Protospacer Adjacent Motif
PAM site is not in the gRNA, it is a little
docking station in the Cas9 protein. It fins a
lot of NGG
options in the
genome.
