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Summary Molecular Biology 1,
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Molecular Biology 1 – Theme 9 – 2020-2021PowerPoint presentations
The function of a gene can be determined or discovered using RNAi, CRISPR, knockouts, mutagenesis
and expression analysis.
CRISPR/CAS is a cheap and easy technique. It can target any
sequence and can be multiplex: targeting more than one sequence in
a cell. The site directed mutagenesis is based on the adaptive
immune system of bacteria (and some archaea).
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
are areas on the genome of bacteria and archaea. When the
bacterium has survived an infection from a bacteriophage, a short
sequence (spacer) of the phage’s DNA is introduced into the genome,
which helps protect from further infection.
When an invading (dsDNA) phage invades the cell, Cas proteins can
recognise a Protospacer Adjacent Motif (PAM), which is NGG or NAG.
The Cas proteins then introduce (part of) the sequence before this
motif into the genome (usually 20 bp). This sequence is called SpacerRNA (=crRNA).
When the phage invades the cell again, the stored sequence is transcribed into RNA. Cas9 binds to
this RNA: processing and hybridisation with trcrRNA (trans activating) and Cas9p. This complex binds
to the matching sequence (from the phage’s DNA) with the PAM sequence behind it. When this
complex binds to the phage’s DNA, a double stranded break is created in the protospacer of the
target DNA.
The spacer sequences are transcribed non-stop but get more expressed when the protein binds to a
phage’s DNA after a second (or third) invasion.
Development of the system lead to people using sgRNA instead of trcrRNA and crRNA.
sgRNA = crRNA + trcrRNA
There are two ways a cell can react to a double stranded break:
- Non-homologous end joining (NHEJ) usually creates indels due to mistakes in the repair
system.
- Homologous recombination (HR) usually adds DNA.
,To avoid stray edits, one can introduce two matches for one break:
- HNH- cuts own strand.
- RuvC- is an endonuclease domain and cuts the complementary strand.
One can also use dCas9: this is a dead or defect Cas9 protein, meaning it will no longer cut the site it
matches with, but simply block it. This will stop transcription of the (viral) genes as well.
In the practical course dCas9 is used. The goal is to stop SunI expression. SunI gives resistance to the
antibiotic SunA, which the bacterium usually produces.
How to obtain a lot of protein (for i.e. research):
1. Isolate plasmid DNA and DNA containing the gene of interest (GOI)
2. Gene inserted into the plasmid (recombinant DNA)
3. Plasmid put into bacterial cell (recombinant bacterium)
4. Cells cloned with GOI
5. Identification of desired clone
6. Various applications: basic research, reinsertion to original organism, etc.
There are (dis)advantages to use certain organisms for overexpression of desired proteins:
- Bacteria and yeast:
o Plus: grow fast and cheap
o Plus: high level of production
o Negative: Post translational modifications are not the same as in a human
- Insect cells:
o Plus: high level of production
o Plus: post translational modifications are possible
- Plants
o Plus: cheap and high production levels
o Plus: good possibilities for purification
o Plus: not always necessary to purify – just eat the plant
- Eukaryotic cell lines
o Plus: works with eukaryotic genes
o Negative: expensive
- Transgenic animals
o Complete organism
, A plasmid is a combination of a replicon, promotor, tag and tag removal sequence, MCS and marker.
A bacterium only has multiple plasmids if they belong to different compatibility groups: they must
have different replication factors.
When a cell has 2 incompatible plasmids, the replication of them stop. As soon as they are
distributed to different daughter cells, plasmid multiplication starts, but only if there is only one
plasmid with the same replication factor, otherwise they will have to wait another replication cycle of
the bacterium.
When there’s competition between the plasmids, the smaller ones are in favour of the bigger ones.
You can add two genes in one plasmid: duetvectors. They have two MCS.
Stringent control: dependent on the presence of initiation proteins synthesized by the host cell.
Relaxed control: can initiate DNA replication independently of the host’s initiation proteins.
The Lac operon is subject to negative control by a repressor.
There are some disadvantages to the Lac operon:
- Leaky: even when turned off, some transcription still takes place. This can be solved by
putting LacI on a plasmid or using a strain with the LacI Q promotor in front of LacI for higher
expression.
- Catabolite repression: even when there is very little glucose in the cell, it will still prefer using
this, even when there is a lot lactose available. By changing two basepairs of LacUV5 in TATA-
box, there is less repression.
The Tac-promot is a combination of the Lac- and Trp-operon. It yields a 10 times higher expression
level than the Lac-operon and 3 times that of the Trp-operon.
The induction happens through either Lac or IPTG.
The T7 promotor consist of pET-vectors. Their target protein is up to 50% of that of total protein.
The gene for phage T7 RNA polymerase is present on an extra plasmid (or in some E. coli strains).
Promotor T7RNAP = LacUV5.
When IPTG or lactose is added, there is an expression of T7RNAP, which then leads to expression of
the gene of interest.
A lysozyme binds T7RNAP to ‘remove’ small bits of T7RNAP. As soon as there is a lot of T7RNAP
formed in the cell, it can induce the gene of interest.
The araPBAD promotor uses pBAD vectors. When arabinose is absent, AraC introduces a loop in the
DNA. When arabinose is present, AraC activates transcription.
This uses positive control (by inducer) and no leakiness.
Phage lambda (=pλ) can enter two life stages: ) can enter two life stages:
- Lytic: new phages are synthesized and leads to cell lysis
- Lysogenic: integrating into host’s genome, to form new phages later.
The phage can change from lysogenic to lytic when there’s DNA damage, food shortage, etc. It
remains lysogenic at low temperatures. It also has a high infection rate (= more Rep = λ) can enter two life stages: cl present).
Repression by λ) can enter two life stages: cl keeps it lysogenic.
DNA damage SOS-response synthesis of RecA RecA cleaves λ) can enter two life stages: cl induction of viral genes.
RecA usually functions as activating genes for DNA-repair.
There are various ways to use pλ) can enter two life stages: promotor.
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