Summary
MBY364 Theme 2 summary
- Course
- MBY364 (MBY364)
- Institution
- University Of Pretoria (UP)
Full summary of theme 2 (plasmids as cloning vehicles) made using lecture notes, the textbook and class notes.
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PLASMIDS AS CLONING VEHICLESBASIC PROPERTIES OF PLASMIDS
- Plasmids are replicons that are stably inherited in an extra-chromosomal state
- They are never integrated into the genome
- Properties
- Genetic homogeneity - identical sequence
- Constant monomeric unit size - they do not become longer/shorter
- Replicates independent from the genomic DNA - has its own oriC
- Exist typically as circular dsDNA
- Both DNA strands intact - covalently closed circular DNA (CCC)
- Only one strand intact - open circle DNA (OC)
- Supercoiled and OC DNA separate upon electrophoresis due to different
structural configurations
- Adding EtBr causes the supercoiled plasmid to unwind
- Adding excess EtBr causes plasmid to rewind in opposite direction
- Widely spread in prokaryotes
- May be 1x106 - 200x106 Da
- May confer a unique phenotypic trait onto their host e.g. antibiotic resistance
or restriction and modification systems
- Cryptic plasmids - no phenotypic traits yet ascribed
- Plasmids can be categorised in one of two major classes
- Conjugative - carry tra genes which promotes conjugation
- Non-conjugative - absence of tra genes
- tra - transfer genes
- Non-conjugative plasmids cannot be introduced using conjugation - use
transformation
- Non-conjugative plasmids are easy to isolate
- Two other forms of plasmids
- Relaxed plasmids - multiple copies per cell
- Stringent plasmids - limited number of copies (1-3) per cell
- Relaxed plasmids have low molecular weights due to no tra genes
- Stringent plasmids contain tra genes
- Importance of the oriC
- Allows replication
- Determines host range in which they replicate
- RP4 - most gram-negative bacteria
- RSF1010 - gram-positive and gram-negative bacteria
- RP4 is on conjugative plasmids and RSF1010 is on non-conjugative plasmids
- Promiscuous - stably maintained in a diverse range of hosts
- This can be bad as the plasmid can transfer antibiotic resistance to many
bacteria
, - Plasmids with a broad host range encode most to all proteins required for
replication
- ColE1 - restricted host range (enteric bacteria)
- ColE1 replicates through an antisense mechanism
- Only a small region is needed for replication - close to oriC
- Large open areas on plasmids are available to be modified - remove plasmid
part and insert foreign DNA
MECHANISMS OF REPLICATION OF PLASMIDS
- Copy number of a plasmid is determined by regulating the initiation of plasmid
replication
- Two major mechanisms:
- Regulation by antisense RNA
- Regulation by binding of essential proteins to repeated sequences
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