Lecture 2: Vectors Flashcards
What are artificial plasmids? What do we put in them?
Plasmids are small extrachromosomal circles of DNA which are covalently closed. They carry genes for antibiotic resistance, conjugation or metabolism of substrates.
• Smaller than natural plasmids.
• Relaxed origin of replication. Needs to be replicated many times. Copy number depends on rate of initiation. Will need a eukaryotic one as well if that is where it is being placed.
• Multicloning site (multiple RE sites). Allow for directional cloning.
• Selectable markers. Antibiotic resistance.
• Need to make sure start codon and ribosome binding site (Shine-Dalgarno sequence are in frame.
• We can also use markers to show if the GOi is inserted. For example, the MCS could be in the lacZ gene. Blue if the GOI is not inserted, white if it is.
What vectors can we use for mammals?
There are two types of transfection which are performed upon mammalian cells:
• Transient transfection: DNA is expressed from an extrachromosomal vector (24-72 hours).
• Stable transfection: continued selective pressure promotes integration. Alternatively, retroviruses could be used.
A mammalian vector often requires a shuttle vector, a vector which can propagate in two different host species.
They often also required other parts:
• Replication origin for bacteria and mammals.
• Selectable markers for bacteria (ampicillin) and mammals (neomycin).
• Expression vectors: rbs, stop codons and promoters (CMV).
• Tag for ease of expression.
What are minicircles?
Minicircles are circles of DNA.
• They begin as part of parental plasmids in bacteria.
• The minicircle is then cut out using site specific recombinases.
• This forms the minicircles and the bacterial parts.
• The resulting minicircle is more efficient as the mammalian cell is less likely to perceive it as foreign.
What are cosmids?
Sometimes we need larger inserts for larger genomes or for when we are creating DNA libraries.
• Cosmids are small (8 kb) but can take larger inserts (around 40 kb).
• Useful for complex genomes in genome mapping projects.
• Use phage lambda cos (cohesion sites).
• Contain plasmid replication origin sites).
• Drug resistance.
• Unique RE sites.
• Can be packaged into lambda phage heads and infect like a phage.
• Circularises when in the cell.
What are fosmids?
Fosmids are similar to cosmids in terms of size and insert capacity.
• Based on F-plasmids (fertility).
• Host cell (E. coli) can only have one fosmid molecule.
• Low copy number means its more stable as there is no recombination.
• Useful for stable libraries from complex genomes.
• Used for checking human genome sequence.
What are artificial chromosomes?
Artificial chromosomes can take huge amounts of DNA (2 Mb). They need a suitable replication origin as they replicate with the host cell genome.
What are bacterial artificial chromosomes?
BACs are circular chromosomes. They are derived from the E. coli F plasmid. They will accept 300 kb of DNA, usually around 100 kbp.
What are yeast artificial chromosomes?
YACs are used in yeast. They can take huge inserts of 2000kb.
• Genes expressed in normal chromosomal context.
• Vector of choice in some genome mapping projects.
• Leucine selectable marker.
• ARS (yeast replication origin).
• Telomeres on each end
• Centromere
• MCS
• YACs often act as shuttle vectors. They are grown in E. coli and cut with REs before foreign DNA is added.
How are human artificial chromosomes made?
HACs are formed spontaneously by adding 3 DNA types to human cells.
1) Telomeric DNA
2) Normal gene containing human DNA
3) Alpha satellite DNA. Non-coding, part of the centreomere.
What are baculoviruses?
Used for eukaryotic protein expression.
• Baculoviruses infect eukaryotic insect cells (e.g. silk worm moth).
• No danger to humans.
• Correct processing.
• Express cDNA from very strong polyhedrin promoter (lots of expression).
• Protein secreted into medium.
• Insert gene into a bacmid (baculovirus shuttle vector) or a polyhedrin gene system in a plasmid vector.
• Use recombinant bacmids to infect cell lines.
• When the viruses infect when a bacmid is present, the gene of interest is inserted.
• Can modify systems to enhance glycosylation.
