Lecture 12 - Genetic Manipulation I Flashcards
genetic manipulation is interpreted as:
the cloning, modification and analysis of DNA sequences
what does genetic manipulation allow us to do?
- produce proteins (i.e: for detailed analysis in structural biology)
- explore the function of genes and pathways (human health)
- identify species and biodiversity
cloning a specific gene allow us to:
- allows specific sequencing of the gene
- allows expression of the protein product in, for example, bacteria
- allows for the modification of a gene to produce an altered protein
the first application of recombinant DNA technology:
bacterial production of human insulin in bacteria (1978 - licensed in 1982)
modifying a whole organisms DNA to explore the consequences allows us to:
- allows analysis of gene function through phenotype of a knockout
- allows the determination of how gene expression is controlled
- allows modification of the gene to produce an altered protein and explore the phenotypic consequences
first inherited modification of a mammalian genome:
1981
examples of genetic manipulation in ecology and biodiversity:
- allows analysis of population movement
- allows identification of species in an environment
- allows the characterisation of microbes that cannot be cultured
an outline of the gene cloning procedure:
(1) the insertion of a fragment of DNA (made through e.g: restriction endonuclease) into a cloning vector (joining: ligation)
(2) the subsequent propagation and amplification of the recombinant DNA molecule in a host organism (each cell grown as a clone)
Cloning allows the ____________ a gene (or other DNA sequence) in a way that allows it to be __________ – for _________ and ____________
(1) purification
(2) replicated
(3) analysis
(4) modification
difference between the purification of an enzyme and a gene:
- enzymes can be purified from cells and their properties can be studied using biochemistry and structural biology, however they can not be replicated and amplified like DNA fragments
why is the cloning of a pure gene sample separated from the mass of an organisms DNA useful?
- allows the DNA sequencing and analysis of features
- allows genetic manipulation in order top change amino acid sequence in a genes subsequent protein
- allows fusion of a gene to a reporter protein such as GFP
- allows gene over-expression for desired protein production
- can help assign structural features of a protein to function
the basics you need to go about cloning a gene:
(1) source of DNA that is to be cloned
(2) a cloning vector to act as the carrier of the DNA being cloned
(3) a way of cutting the source DNA into appropriate size (usually a restriction enzyme) and linearizing the vector
(4) a method for joining DNA fragments together
(5) a method for joining DNA into E.coli cells
cloning vector:
a DNA molecule, capable of replication in a host organism, into which a DNA sequence [gene] is inserted to construct a recombinant DNA molecule
common features of cloning vectors:
- replicate independently of and at a higher level than the host chromosome: aiding purification
- contain a selectable marker (e.g: antibiotic resistance gene) - allowing fore identification and selection of vector containing host bacteria
- contain specific restriction enzyme recognition sites (cloning sites) allowing linearisation of circular DNA sequences into the vector
- usually derived from naturally-occuring plasmids and bacteriophages
most common vectors:
bacterial plasmids
characteristics of natural plasmids:
- extrachromosomal
- mostly circular (some linear)
- 1kb-100kb in size
- non-essential
- usually carry ‘supplemental’ genetic information, but may be cryptic
- often confer selective advantage on the host bacterium
antibiotic resistance plasmids code for:
- encode for proteins that provide resistance to antibiotics
sex plasmids allow for:
sex plasmids allow for the exchange of DNA between bacteria
function of col plasmids:
col plasmids encode for various toxins that kill neighbouring cells
degradative plasmids:
degradative plasmids encode for a variety of enzymes that allow the metabolism of unusual substances
virulence plasmids:
virulence plasmids generally encode proteins that turn a bacterium into a pathogen
characteristics of bacteriophages as vectors:
- can be either circular of linear DNA
- 5kb to hundreds of kb in length
- infect the host, replicate and package DNA, then kill the cell
- some viruses can integrate into the host chromosome and lay dormant
plasmid cloning vectors have been engineered so that they are small and contain (+ what do they also sometimes contain?):
- a replication origin
- a selectable marker
- suitable cloning site(s)
sometimes they also contain:
- a multiple cloning site (MCS)
- a method for detecting recombinant molecules
the selectable marker:
- usually an antibiotic resistance gene such as the b-lactamase gene (ampicillin resistance)
examples of plasmids (1) - pBR327:
- replication of origin (Ori): derived from natural plasmids of the “ColE1” family, promotes replication initiation by host replication enzymes
- restriction enzyme sites: engineered to contain several unique sites including Pstl, EcoRI, HindIII, Al/I - which can all be used for cloning
- has two selectable markers derived from native plasmids: (1) ApR (ampicillin resistance) (2) TcR (tetracycline resistance)
examples of plasmids (2) - pBluescript SK+:
- ampicillin resistance gene
- origin of replication is deregulated for high copy number (500/cell)
- lac promoter and lacZa gene
- MCS multiple coding sites
- bacteriophages origin to make ssDNA (doesn’t have other proteins for phage production
restriction enzymes:
- usually cut a palindromic (e.g symmetrical) sequence
- derived from a wide variety of (usually) bacterial sources
- can generate ‘sticky’ ends which are resealable with DNA ligaments: [3’ OH] & [5’ Phosphate]
in what two ways can restriction enzymes cut?
restriction enzymes can cut both sticky and blunt ends
different restriciton enzymes sometimes produce the same sticky ends:
this means that certain fragments can be legates into vectors that are digested with BamHI - something that is particularly useful when making a genomic library
in some instances this means that the resulting hybrid site can generally not be cut again
origin of restriction enzymes:
- mainly derived from bacteria that are protecting themselves from viral invasion
- a some of the “rare cutters” (i.e: >8 base cutters) are derived from single cell eukaryotes and are involved in various aspects of biology such as mating types in yeast
why is bacterial DNA not degraded?
this is because bacterial DNA is often methylated
insertional inactivation: lacZa and Blue-white screening:
•The b-galactosidase is a large enzyme encoded by lacZ (~1000 amino acids)
•LacZ enzyme can be split into 2 peptides – a small fragment called LacZa and large fragment called LacZW
•Neither LacZa or LacZW are active alone – BUT they can combine to form active enzyme
•Introduction of a plasmid expressing LacZa into a strain that already expresses LacZW will lead to functional b-galactosidase activity
•Active b-galactosidase can convert the colourless substrate X-gal into a BLUE product
natural “transformation”:
- bacteria can take up DNA from the environment, usually from other dying organisms
- some bacteria are naturally competent (i.e: can constitutively take up DNA)
- some bacteria are inducibly competent. This means they can become competent under particular growth conditions such as stress
hypotheses to why cells competent:
- H1: a primitive form of “sex” to aid gene diversity
- H2: DNA as a food, simply using resources available
- H3: helps to repair DNA, particularly under stressed conditions
- H4: allows horizontal gene transfer which gives an evolutionary advantage
making E.coli cells competent artificially:
- E.coli cells are grown to mid-log phase
- they are harvested by centrifugation
- then they are incubated in ice cold CaCl2
this results in the cells to take up DNA
