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pBR322 Vector : pBR322 is a plasmid and was the first widely-used E. coli cloning vectors. Created in 1977 in the laboratory of Herbert Boyer at the University of California San Francisco, it was named after the Mexican postdoctoral researchers who constructed it. The p stands for "plasmid," and BR for "Bolivar" and "Rodriguez." pBR322 is 4361 base pairs in length and contains the replicon of plasmid pMB1, the ampR gene, encoding the ampicillin resistance protein (source plasmid RSF2124) and the tetR gene, encoding the tetracycline resistance protein (source plasmid pSC101). The plasmid has unique restriction sites for more than forty restriction enzymes. 11 of these 40 sites lie within the tetR gene. There are 2 sites for restriction enzymes HindIII and ClaI within the promoter of the tetR gene. There are 6 key restriction sites inside the ampR gene. The origin of replication or ori site in this plasmid is pMB1 (a close relative of ColE1). The ori encodes two RNAs (RNAI and RNAII) and one protein (called Rom or Rop ). The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the count increases through the tet genes. The ampicillin resistance gene is a penicillin beta-lactamase. Promoters P1 and P3 are for the beta-lactamase gene. P3 is the natural promoter, and P1 is artificially created by the ligation of two different DNA fragments to create pBR322. P2 is in the same region as P1, but it is on the opposite strand and initiates transcription in the direction of the tetracycline resistance gene 2. Limitations of Eukarotic gene expression in prokaryotic host 1. no splicing of introns/exons ,need cDNAs for eukaryotic genes 2. foreign promoters do not work in E. coli (unless from closely related Gram negative bacteria) – cDNAs do not have promoters, need promoter from E. coli gene (–35, –10 sequences) – and bacterial ribosome binding site e.g. lac promoter – controlled by LacI repressor, lacIq overexpresses repressor stops expression of toxic protein until induced by IPTG but it is not suitable for large scale (industrial) also controlled by catabolite repression glucose in media reduces cyclic AMP (cAMP) levels in cell inactivates cAMP activator protein (CAP) cAMP-bound form needed for expression from lac promoter expression from lac promoter only in media without glucose trp promoter – from tryptophan biosynthesis operon controlled by tryptophan in media induced under low tryptophan conditions stronger promoter than lac & has better ribosome binding site tac or trc promoters (1 bp difference) have –10 sequence from lac promoter controlled by IPTG have –35 sequence from trp promoter no catabolite repression 3. proteases may degrade foreign proteins e.g. Lon protease degrades denatured/misfolded proteins including many foreign proteins , clone in lon mutant strains 4. proteins may form inclusion bodies insoluble aggregates of protein denatured proteins form advantage: – easy to purify ,lyse cells and pellet aggregates by centrifugation problems: – must denature proteins to solubilize urea, detergents often hard to refold into active form promoted by: large size of polypeptide chain more common if polypeptides >100 amino acids best expression results with smaller peptides e.g. insulin, human growth hormone large amounts of protein high copy number vectors, strong promoters high temperatures (40°C) – denature proteins usion proteins – may solve problems 2, 3, &/or 4 at once from vectors with 5' ends of E. coli genes multiple cloning site in gene The concept of alpha-complementation is important because it is a quick, easy, 1-step process of determining whether a transformed bacterial colony has plasmid+insert or not (The tetracycline resistance of pBR322 works, it is just time consuming). The key to alpha-complementation is the fact that the lac-Z gene product (B-galactosidase) is a tetramer, and each monomer is made of two parts - lacZ-alpha, and lacZ-omega. Researchers determined that if the alpha fragment was deleted, the omega fragment is non-functional; however, alpha fragment functionality can be restored in-trans via plasmid. Hence, then name alpha-complementation. What is needed for this to work as a cloning technique is a strain of E. coli that has the deletion of the lac Z-alpha (lacZ DM15 works well as a genotype), and a plasmid with the lacZ-alpha fragment as the scorable marker (such as pBluescript or pCR2.1). If plain plasmid is successfully transformed into a cell, then the cell will express functional B-galactosidase. However, if the plasmid+insert is transformed into a cell, then it will express non-functional B-galactosidase (the lac Z-alpha will be disrupted with the insert gene product). Plate the cells out onto selection media based on the selectable marker, IPTG (induces lac repressor to disengage), and X-gal (chromogenic substrate that yields blue product when cleaved by B-galactosidase) and the white colonies (non-functional B-galactosidase) are the ones with plasmid + insert; the blue ones have plain plasmid. Another method of accomplishing the same task is to completely delete the lac operon from the chromosome, but introduce lac-Z-omega fragment on an F’ fertility factor. This accomplishes the same task as above, with a plasmid with lac-Z-alpha scorable marker. BAC: A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome's usual insert size is 150-350 kbp. A similar cloning vector called a PAC has also been produced from the bacterial P1-plasmid. BACs are often used to sequence the genome of organisms in genome projects, for example the Human Genome Project. A short piece of the organism's DNA is amplified as an insert in BACs, and then sequenced. Finally, the sequenced parts are rearranged in silico, resulting in the genomic sequence of the organism. 1. oriS, repE – F:for plasmid replication and regulation of copy number. 2. parA and parB: for partitioning F plasmid DNA to daughter cells during division and ensures 3. A selectable marker for antibiotic resistance; some BACs also have lacZ at the cloning site for 4. T7 & Sp6 phage promoters for transcription of inserted genes. 2Yeast artificial chromosome (YAC): is a vector used to clone DNA fragments larger than 100 kb and up to 3000 kb. YACs are useful for the physical mapping of complex genomes and for the cloning of large genes. First described in 1983 by Murray and Szostak, a YAC is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences named autonomous replicating sequence needed for replication and preservation in yeast cells. A YAC is built using an initial circular plasmid, which is typically broken into two linear molecules using restriction enzymes; DNA ligase is then used to ligate a sequence or gene of interest between the two linear molecules, forming a single large linear piece of DNA. A typical YAC consists of centromere element (CEN) for chromosome segregation during cell division, telomere and origin of replication (ori) were isolated and joined on plasmid constructed in E.coli. For cloning purpose YAC is digested with restriction enzymes and recombinants are produced by inserting a large fragment of genomic DNA. This molecule can be maintained in yeast as YAC. The transformant that contain YAC can be identified by red/white color selection. Non-transformed yeast contain white colonies. Red colonies of yeast contain recombinant YAC molecule. Due to insertion of DNA molecule into EcoR1 site, SUP4 gene is inactivated and no proteins will be expressed . This facilitates to develop red colonies. A phagemid or phasmid is a type of cloning vector developed as a hybrid of the filamentous phage M13 and plasmids to produce a vector that can grow as a plasmid, and also be packaged as single stranded DNA in viral particles. Phagemids contain an origin of replication (ori) for double stranded replication, as well as an f1 ori to enable single stranded replication and packaging into phage particles. Many commonly used plasmids contain an f1 ori and are thus phagemids. Similarly to a plasmid, a phagemid can be used to clone DNA fragments and be introduced into a bacterial host by a range of techniques (transformation, electroporation).

Source: http://www.gla.ac.in/glau/download/rdt%20ii%20_msb-124_.pdf