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Summary BMS75 - Advanced Tools in Molecular Biology
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Summary BMS75 - Advanced Tools in Molecular Biology including lectures and an E-learning on Cloning methods.
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Renske de Veer (rdeveer)BMS#75 Advanced tools in molecular biology
Lecture 1. Introducing the master course and the use of molecular probes in cell adhesion
and migration.
Molecular tools in cell biology
- To visualize protein expression (spatio-temporal)
o Tagging protein
▪ Biochemical or fluorescent tag for example
▪ Determine localization
o Tagging peptides with a role in compartmentalization
o Expressing fluorescent cell cycle proteins
- To examine protein by
o Changing protein amounts (overexpression, downregulation → siRNA, shRNA,
Crispr/Cas)
o Perturbing protein function (loss of function- or activating mutations)
o Applying function sensors.
Nucleus, intermediate filament, microfilament, smooth endoplasmic reticulum and associated
proteins are coupled to GFP or RFP → during practicum.
Molecular tools used in current research
- Localization proteins:
o Actin-based structures in cells
▪ Actin-myosin based contraction.
- Molecular therapy in myotonic dystrophy
o Expanded DM genes in myotonic dystrophy: elimination of (C/CUGn) RNA by Crispr-
Cas9.
Learing trajectory
- Basics, vector design: theoretically.
,Renske de Veer (rdeveer)
Lecture 2. Designing molecular probes.
Central dogma
- Extracellular proteins are translated in the ER. These are exocytoses at one point via golgi.
- Transmembrane proteins are translated in ribosome.
DNA replication:
- One strand is easily translated from 5’→ 3’, the other end is more difficult because reading
frame from 3’ → 5’. Results in okazaki fragments (small DNA fragments). Connecting these
fragments ligases are necessary.
GAP prevents 5’ degradation of mRNA, poly-a-tail provides stability to mRNA molecules (happens
post-transcriptionally).
Splicing out of introns happens post-transcriptionally.
Open reading frame/coding sequence: region between ATG (start codon: AUG) and stop codons.
In bacteria and viruses: multiple ribosome-binding sites.
Mature RNA has non-coding sequences.
In nucleus all introns are spliced out from mRNA, mRNA is transported out of the nucleus with
untranslated 5’ and 3’ regions.
Reading frame: coding of triplets.
Protein routing:
1. Transport through nuclear pores
2. Transport across membranes
3. Transport by vesicles.
- Routing signals:
o NLS, NES
o Signal peptide
o Transmembrane domain
o Mannose-6-P
o GPI anchors, lipid tails
o Amphipathic helix
o SKL
RNA flavours
- Types of RNA produced in cells
o mRNAs: code for proteins
o rRNA: form the core of the ribosome and catalyse protein synthesis (ribozyme)
o miRNAs: regulate gene expression
o tRNAs: serve as adaptors between mRNA and amino acids during protein synthesis
o Other small RNAs: used in RNA splicing, telomere maintenance, and many other
processes
- Three RNA polymerases in eukaryotic cells
o RNA polymerase I: most rRNA genes
o RNA polymerase II: protein-coding genes, miRNA genes, plus genes for some small
RNAs
o RNA polymerase III: tRNA genes, 55 rRNA gene, genes for many other small RNAs.
, Renske de Veer (rdeveer)
Definition of cloning:
- Cutting a piece of DNA from one organism and inserting it into a vector where it can be
replicated by a host organism (also called subcloning).
o Agents used:
▪ Plasmids (non-genome wide cloning, non-library)
• Not found in mammalian cells.
• Plasmids are circular pieces of double stranded DNA found naturally
in bacteria
• Plasmids can carry antibiotic resistance genes, genes for receptors,
toxins or other proteins
• Plasmids replicate separately from the genome of the organism
• Plasmids can be engineered to be useful cloning vectors (vector
(never use vector for naturally occurring plasmid)= plasmid used for
genetic engineering).
• Usually work with max 11/12 kb size of plasmid (/vector), naturally
occurring plasmids are much larger.
▪ Bacteriophages (library)
• Phage Lambda is a bacteriophage/phage (i.e. bacterial virus that uses
e.coli as host)
• Its structure is that of a typical phage: head, tail, tail fibres
o Head contains the DNA, which is inserted through the tail.
Physical size of head limits amount of DNA that can be
stored in it. In order for DNA to fold it into the head cos-
sequences are used.
o Binding proteins are bound to tail fibres
• The bacteriophages uses for cloning are the phage Lambda and M13,
it affects bacteria
• Follow either lytic or lysogenic cycle
o Lytic cycle: harmful cycle
• There are two kinds of lambda phage vectors: insertion vector and
replacement vector
• Insertion vectors contain a unique cleavage site whereby foreign
DNA with size of 5-11 kb can be inserted.
▪ Cosmid (library) → derivative of both phages and plasmid.
• A cosmid a s type of hybrid plasmid that contains a Lambda phage
cos sequence
• Cosmids are plasmids that incorporate a segment of bacteriophage
Lambda DNA that hase the cohesive end site (cos) which contains
elements required for packaging DNA into Lambda particles. It is
normally used to clone large DNA fragments between 28-45 kb
• Cosmid can replicate in bacterial cell, so infected cells grow into
normal colonies
• Insert DNA limited by the amount of DNA that can fit into phage
capsule
• Somewhat unstable, difficult to maintain
▪ Artificial chromosome vectors (library)
• Linear or circular
• 1 or 2 copies per cell
• Different types:
o Bacterial → used for human genome project
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