plasmids and vectors - Instructor Supplement to pGlo Bacterial Transformation

Plasmids and Vectors Instructor Supplement to pGlo Bacterial Transformation A more detailed look at plasmids Origin of Replication Multiple Cloning Site Promotor Site Antibiotic Resistance Gene Cloning into a Plasmid  People believed that “safe” strains of bacteria, viruses and vectors could be made in a few weeks  NIH formed the Recombinant DNA Advisory Committee (RAC)  It took 1 year (1976) before the first “safe” (EK2 category) line of E. coli was released  That year, RAC released a set of guidelines requiring the use of safe bacteria Asilomar Conference NIH Guidelines  Self Regulation in Science Milestone  Contents  Specified handling and construction processes  Microorganisms containing recombinant DNA were prohibited outside of the laboratory  Vectors that sexually move to “unsafe” bacteria was prohibited  Subsequent modifications  1986 expanded to include animals and plants, and 4 biosafety levels  1994 officially relinquished control of GMO plants in the environment to EPA and APHIS The First “Safe” Bacterium  Released in 1976 by Roy Curtiss III at the University of Alabama  E. coli χ1776  Required diaminopimelic acid (DAP)  Fragile cell walls (low salt, detergent sensitive)  Difficult to work with  Slow grower  Poor receptor for transformation In the 1970’s and 1980’s  The first cloning vectors such as pSC101 had limited functionality  The next trend was to develop smaller plasmids  Advantages  Increased efficiency of transformation  Easier to restriction map  Higher copy numbers The Cadillac of Cloning Vectors  pBR322  Clone fragment in one antibiotic gene  Select for other antibiotic resistance  Screen for presence of one resistance gene (selects against untransformed bacteria) and loss of resistance to interrupted antibiotic resistance gene (selects for recombinant molecule) pBR322 4,361 bp EcoRI Tet R Amp R APstI BamHI Screening bacteria by replica plating [...]... into restriction site, the cosmid packaged into viral particles and these phages used to infect E.coli Cosmid can replicate in bacterial cell, so infected cells grow into normal colonies Insert DNA limited by the amount of DNA that can fit into phage capsule Somewhat unstable, difficult to maintain ori TetR 21.5 kb cos EcoRI Cos site is the only requirement for packaging into phage particle Other Vectors. .. Vectors  BACs (Bacterial artificial chromosomes)     YAC (Yeast Artificial Chromosome)      Large low copy number plasmids (have ori and selectable marker) Can be electroporated into E coli Useful for sequencing genomes, because insert size 100 - 300kb Can be grown in E.coli and Yeast Miniature chromosome (contains ori, selectable markers, two telomeres, and a centromere Can accept 200 kb -1 000... Can accept 200 kb -1 000 kb; useful for sequencing Ti plasmids; to introduce genes into plants Expression vectors How do you identify and clone a gene of interest?  Screen A DNA library:    Genomic cDNA Use Polymerase Chain Reaction (PCR) to clone gene of interest Genomic Library 25 cDNA library What can you do with a library?   Can be used to complement a mutant (this is more common for research... polylinkers into plasmid vectors Polylinker is a tandem array of restriction endonuclease sites in a very short expanse of DNA For example, pUC18’s polylinker  Sites for 13 RE’s  Region spans the equivalent of 20 amino acids or 60 nucleotides Source: Bio-Rad Laboratories The Polylinker Advantage    Unique sites (usually) Insert excision facilitated Restriction endonuclease mapping and Subcloning... and Subcloning made easier Another Major Advance: Blue-White Screening Features of many modern Plasmids •Small size •Origin of replication •Multiple cloning site (MCS) •Selectable marker genes •Some are expression vectors and have sequences that allow RNA polymerase to transcribe genes •DNA sequencing primers The Major Limitation of Cloning in Plasmids  Upper limit for clone DNA size is 12 kb  Requires... non-essential parts of lambda Can now insert large pieces of DNA (~ 20 kb) Lysis Replication ori COS Head Tail Lambda was great:   Larger insert size Introducing phage DNA into E.coli by phage infection is much more efficient than transforming E.coli with plasmid DNA But:  Have to work with plaques Cosmids      Hybrid vectors: plasmids that contain bacteriophage lambda cos sites DNA (~ 3 3-4 8... overlapping regions needed to place in proper sequence Preference for smaller clones to be transformed If it is an expression vector there are often limitations regarding eukaryotic protein expression   Bacteriophage lambda (λ) A virus that infects bacteria o In 1971 Alan Campbell showed that the central third of the genome was not required for lytic growth People started to replace it with E coli