1. Plasmids and Vectors Instructor Supplement to pGlo Bacterial Transformation

2. A more detailed look at plasmids Origin of Replication Multiple Cloning Site Promotor Site Antibiotic Resistance Gene

3. Cloning into a Plasmid

5. 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

6. 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

7. The First “Safe” Bacterium  Released in 1976 by Roy Curtiss III at the University of Alabama  E. coli 1776 Required diaminopimelic acid (DAP) Fragile cell walls (low salt, detergent sensitive) Difficult to work with Slow grower Poor receptor for transformation

8. 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

9. 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

10. Screening bacteria by replica plating

11. Next Major Advance in Plasmid(ology)  The inclusion of 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

12. The Polylinker Advantage  Unique sites (usually)  Insert excision facilitated  Restriction endonuclease mapping and Subcloning made easier

13. Another Major Advance: Blue-White Screening

14. 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 Features of many modern Plasmids

15. The Major Limitation of Cloning in Plasmids  Upper limit for clone DNA size is 12 kb  Requires the preparation of “competent” host cells  Inefficient for generating genomic libraries as 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

16. Bacteriophage lambda (λ) o 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 DNA

17. Lambda genome is approximately 49 kb in length. Only 30 kb is required for lytic growth. Thus, one could clone 19 kb of “foreign” DNA. Packaging efficiency 78%- 100% of the lambda genome. A complete animation of the lytic cycle: http://www.blackwellpublishing.com/trun/artwork/Animations/Lambda/lambda.html

18. Bacteriophage lambda  Protein capsule of lambda has a tight constraint on the amount of DNA that will fit inside it (~ 55kb)  By the early 1970’s we knew that a good portion of lambda was not required  “Junk” DNA COS site: Cohesive “sticky” ends Lysis Lysogeny Head Tail Replication Circularized lambda ori

19. Not Quite Bacteriophage lambda  Eliminate the non-essential parts of lambda  Can now insert large pieces of DNA (~ 20 kb) COS Lysis Head Tail Replication ori

21. 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  Have to work with plaques But:

22.  Hybrid vectors: plasmids that contain bacteriophage lambda cos sites  DNA (~ 33-48 kb) cloned 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 cos Tet R EcoRI 21.5 kb ori Cos site is the only requirement for packaging into phage particle Cosmids

23. Other Vectors  BACs (Bacterial artificial chromosomes) 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  YAC (Yeast Artificial Chromosome) Can be grown in E.coli and Yeast Miniature chromosome (contains ori, selectable markers, two telomeres, and a centromere Can accept 200 kb -1000 kb; useful for sequencing  Ti plasmids; to introduce genes into plants  Expression vectors

24. 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

25. 25 Genomic Library

26. cDNA library

27. What can you do with a library?  Can be used to complement a mutant (this is more common for research in bacteria).  Can use it in a colony hybridization.

28. Screening libraries by colony hybridization

29. Polymerase Chain Reaction (PCR)

31. Agarose gel electrophoresis

32. Restriction Mapping