Vectors for Cloning - Lecture 3 PDF

ECTORS FOR GENE CLONING Can replicate independently Some plasmids can integrate into the host...

ECTORS FOR GENE CLONING Can replicate independently Some plasmids can integrate into the host chromosome and can replicate through multiple cell divisions – episomes or integrative plasmids Ideally small to facilitate isolation and handling Supercoiled DNA e as e er as endonuclease om r gy is A po e ase Open N nu c l to Relaxed D endo circular (covalently A li gase DNA closed circular DN DNA)  Desired range The host range of a plasmid is determined by the plasmid-encoded proteins that are located close to the ori (origin of replication) region. Plasmids with ori derived from Col E1 can only replicate in enteric bacteria (E.coli, salmonella, etc). RP4 will replicate in most Gram negative bacteria Copy number: number of molecules of an individual plasmid in a single bacterial cell 50 STRINGENT RELAXED The copy number of a plasmid is determined by regulating the initiation of plasmid replication Mechanisms controlling copy number: 1. Regulation by antisense RNA (in Col E1 type plasmids) RNA II (555 base) RNA II acts as a primer only when cleaved by RNase H to leave a free 3’-OH group  RNA I (108 base) encoded by the same region of DNA as RNA II but by the complementary strand can hybridize to form dsRNA helix which cannot be processed by RNase H. High conc of plasmid  high conc of RNA I  inhibition of plasmid replication Rop enhances pairing between RNA I and RNA II and thus inhibits replication even at low concentrations of RNA I Deletion of ROP gene or mutations in RNA I result in increased copy numbers opy number regulation by iterons Iterons are repeating DNA sequences in the ori: 3-7 copies of an iteron sequence which is 17-22 bp long Ex. pSC101 has 3 iteron sequences R1, R2 and R3 Plasmid P1 has 14 iterons shown below: Papp et al., (1994) 3 main functional units: 1. The origin (ori) The only plasmid-encoded protein 2. The initiator gene (repA) required for replication. It binds to iterons and initiates DNA synthesis 3. The copy number control locus (incA) opy number control in pSC101 As RepA concentration drops, replication will be initiated RepA represses its own synthesis by binding to its own promoter region and blocking transcription of its own gene High copy number means synthesis of RepA is suppressed. At higher concentrations, RepA protein can link two plasmids together by binding to their iteron sequences and preventing them from initiating replication (“handcuffing”) REPLICATION OF ITERON PLASMIDS DEPENDS BOTH ON THE CONCENTRATION OF RepA PROTEIN AND ON THE CONCENTRATION OF THE PLASMIDS AS WELL. tioning of plasmids during cell division – segregation. egation instability is the loss of plasmids due to defective partitioning. Ensures plasmids are stably maintained at each cell division pSC101 Genetic structure of the par locus Par from plasmid R1. operon parC is a centromere-like region that promote contains the par promoter and 10 r (320 codons) (117 codons) direct repeats (indicated by small repeated arrows) to which ParR binds. ParR thereby autoregulates transcription of the par operon. similarity to analogous regions present in all proteins with the actin fold Model for R1 par-mediated plasmid partitioning during the cell cycle. 1. Plasmids (red) are replicated by the host cell replication machinery (yellow), which is located at mid- cell (Lemon and Grossman, 1998; Koppes et al., 1999). 2. Replicated plasmid parC regions are paired through interactions with ParR protein (grey), thereby forming a partitioning complex. 3. The partitioning complex forms a nucleation point for ParM filamentation. Continuous addition of ATP-ParM (green) to the filament poles provides the force for active movement of plasmid copies to opposite poles. 4. Within the filaments, ATP is hydrolysed, leading to destabilization of the ParM polymer. Nucleotide exchange is required to recharge the ADP-ParM (blue) molecules for a subsequent round of partitioning. par-/- cells show decreased overall superhelical density compared to WT cells Partition-defective mutants of pSC101 can be stabilized with topA mutation, which increases negative DNA supercoiling Inhibitors of DNA gyrase or mutations in DNA gyrase increases the rate of loss of par-defective pSC101. More than one kind of plasmid can exist in the same cell: compatible Plasmid incompatibility is the inability of two different plasmids to coexist in the same cell in the absence of selection pressure (30 incompatibility groups in E.coli). Plasmids will be incompatible if they have the same mechanism of  Conjugation and transfer are controlled by tra (transfer) and mob (mobilizing) genes It is not yet proven if plasmid transfer can occur only through the pilus Often times, non-conjugative plasmids can also get transferred together with conugative plasmids if their mob region is functional Based on the main characteristics coded by the plasmid genes, they can be classified as 1. F (fertility) plasmids carry tra genes to promote conjugal transfer of plasmids 2. R (resistance) plasmids carry genes conferring resistance against anti-bacterial agents (chloramphenicol, ampicillin, mercury, etc.) 3. Col plasmids code for colicins that kill other bacteria (ex. ColE1 of E.coli) 4. Degradative plasmids allow bacteria to metabolize unusual molecules such as toluene (ex. TOL in Pseudomonas putida) and salicylic acid 5. Virulence plasmids confer pathogenicity on the host bacterium (ex. Ti plasmids of Agrobacterium tumefaciens which induces crown gall disease in plants. Pseudomonas sp Eukaryotic plasmids The yeast 2 µm plasmid (in Saccharomyces cerevisiae) is so far one of the few known eukaryotic plasmids.  irable features of a plasmid for cloning: Low molecular weight Ability to confer readily selectable phenotypic traits on host cells Single sites for a large number of restriction endonucleases herefore an ideal vector should have the following:. An origin of replication. A selectable marker. Suitable single restriction sites. Suitable size. High copy number 6. Insertional inactivation mechanism. Disablement Plasmids are named after the workers who isolated or constructed them: pBR322: developed by Francisco Bolivar and Rodriguez Ampicillin blocks the cross- linking of polysaccharide side chains in the bacterial cell wall, thus eventually lysing the cells. ampR encodes β-lactamase that hydrolyses the β-lactam ring of ampicillin thus inactivating it. Early scientists used naturally occuring Col E1 or pSC101 but their application was limite Improvement up on the construction of pBR322 vectors: pBR325 encodes chloramphenicol resistance in addition to ApR and TcR with a unique EcoRI site in the CmR gene. pBR327 obtained by deletion of a 1089bp segment within pBR322 (deletion derivative) enabling higher copy number and making it non- conjugative Newer construction of vectors was simplified with the use of polylinkers or multiple cloning sites (MCS) pUC19 has a 2.8kb MCS containing sites for many RE thus increasing the number of cloning strategies available. The MCS is inserted into the lacZ’ sequence which encodes the promoter and alpha-peptide of betagalactosidase.  β-Galactosidase is normally coded by the gene lacZ, which resides on the E. coli chromosome. Some strains of E. coli lacks the segment (lacZ’) that codes for the α-peptide portion of β-galactosidase). These mutants can synthesize the enzyme only when they harbor a plasmid (pUC8) that carries the missing lacZ’ segment. α-complementation isopropyl-b-D-thiogalactoside (IPTG) (inducer but not a substrate) 5-bromo-4-chloro-3-indolyl-b-D- galactoside (X-gal) Lac selection (AmpR β-gal+) (AmpR β-gal-) Advantages of using the pUC8 vectors: 1. Chance mutation in the ori turned the plasmid into a high copy number (500-700 before amplification) 2. Identification of recombinant cells in a single step 3. Clustering of restriction sites in the MCS allowing different sticky ends