Plasmids(microbiology)

description of plasmids and types and importance of plasmids and artificial plasmids(PBR322,cosmids,phagemids) and selection of the recombinants and uses and advantages and disadvantages of the plasmids

1. PLASMIDS D.INDRAJA

2. What is? A plasmid is a small, circular, double-stranded DNA molecule that is distinct from a cell's chromosomal DNA It replicates independently of chromosomal DNA Plasmids naturally exist in bacterial cells, and they also occur in some eukaryotes. plasmids carry genes that are beneficial to the host organisms. Often, the genes carried in plasmids provide bacteria with genetic advantages, such as antibiotic resistance. Plasmids have a wide range of lengths, from roughly one thousand DNA base pairs to hundreds of thousands of base pairs. When a bacterium divides, all of the plasmids contained within the cell are copied such that each daughter cell receives a copy of each plasmid. Bacteria can also transfer plasmids to one another through a process called conjugation

3. FEATURES OF PLASMID .

4. ADVANTAGE Scientists have taken advantage of plasmids to use them as tools to clone, transfer, and manipulate genes. Plasmids that are used experimentally for these purposes are called vectors. Researchers can insert DNA fragments or genes into a plasmid vector, creating a so-called recombinant plasmid . Then, because bacteria divide rapidly, they can be used as factories to copy DNA fragments in large quantities .  Artificial plasmids are widely used as  vectors  in  molecular cloning , serving to drive the replication of  recombinant DNA  sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via  transformation .   Characteristics such as flexibility, versatility, safety, and cost-effectiveness enable molecular biologists to broadly utilize plasmids across a wide range of applications. Some common plasmid types include cloning plasmids,  expression plasmids , gene knock-down plasmids, reporter plasmids,  viral plasmids , and  genome engineering  plasmids.

5. Types of Plasmids There are five main types of plasmids: fertility F-plasmids resistance plasmid virulence plasmids degradative plasmids Col plasmids.

6. ARTIFICIAL PLASMID pBR322 is a purpose built plasmid vector and was one of the first widely used cloning vector it has a relatively small size of 4361 bp . This is important because transformation efficiency is inversly proportional to size. Nomenclature of pBR322 Created in 1977 in the laboratory of Herbert Boyer at the university of california , san Francisco ‘p’  plasmid ‘BR’  Boliver and Rodriguez (researchers who developed it) ‘322’  distinguishes those plasmids from others (like pBR325,pBR327 etc) developed in the same laboratory Features of pBR322 pBR322 has 4361 base pairs in length It has two antibiotic resistance genes they are: a) Ampicillin resistance gene( Amp R ) b) Tetracyclin resistance gene ( Tet R )

7. Origin of replication( ori ) It has unique restriction sites for more than 40 restriction enzymes.11 of these 40 sites lie with in the Tet R gene. There are 2 sites for restriction enzymes HindIII and claI with in the promoter of Tet R gene. They are 6 restriction sites inside the Amp R gene. Construction of pBR322 The amp R gene originally resided on the plasmid RSF2124(a naturally occurring antibiotic resistance plasmid in E.coli ) The tet R is derived from Psc101 (a second antibiotic resistant plasmid)

8. The origin of replication is derived from pMB1, which is closely related to the colicin producing plasmid colE1 INSERTIONAL INACTIVATION It is a technique used in recombinant DNA technology to select recombinant colonies It involves the inactivation of a gene up on insertion of another gene inside in its place or with in its coding sequence For example-pBR322 is opened by using a restriction enzyme BamHI where restriction site lies with in the tetracycline resistant gene. The foreign DNA also isolated with the same type of restriction enzyme and DNA ligase is added to the mixture containing linearized pBR322 and the foreign gene two types of vectors are formed Recombinant vector  foreign gene Un altered vector  foreign gene

9. When this mixture of both altered(recombinant)and unaltered vector are used for transformation of E.coli two types of cells may be formed Non Transformed cells Transformed cells Both the transformed cells are transferred to ampicillin containing media then Transformed cell live Non transformed cell  dead Now the only transformed cells are transferred to ampicillin containing media and this is considerd as master plate To identify recombinant cells replicate plate is prepared containing tetracycline media in this Transformed with recombinant  dead as insertional inactivation happened Transformed with non recombinant  live as it is resistant to both antibiotics Now by comparing with the master plate recombinat transformed cells are identified With unaltered vector With recombinant vector

10. amp R tet R Foreign gene amp R amp R tet R Insertion inactivation Unaltered vector Altered vector E.coli Non transformed cell Transformed cells with and with Out recombinant vector INTERPRETATION Non transformed  amp S ,tet S T.With nonrecombinant  amp R ,tet R T,with recombinant  amp R ,tet S Transformation

11. Non transformed cell Transformed cells with and with Out recombinant vector Amp Non transformed cells  dead Transformed cells live Amp Transformed cells are transferred Master plate tet Replica plate Transformed with recombinant  dead Transformed with unaltered  live Compared with the master plate and recombinants are picked INTERPRETATION Non transformed  amp S ,tet S T.With nonrecombinant  amp R ,tet R T,with recombinant  amp R ,tet S

12. USES It is widely used as a cloning vector It is used as a model system for study of prokaryotic transcription and translation ADVANTAGES Due to small size enables easy purification and manipulation As due to the presence of 2 selectable markers ( amp,tet ) allow easy selection of recombinant DNA It can be amplified up to 1000-3000 copies DISADVANTAGES It has very high mobility that is it can move to another cell in the presence of conjugative plasmid like F-cell.due to this the vector may lost in the mixed host of cells There is a limitation in the size of gene of interest that it can accommodate Screening process is time consuming and laborious No high copy number

13. PHAGEMID

14. These are artificially constructed Phagemids are the vectors that are made up of a plasmid and phage DNA Nomenclature of phagemid Phage  part of DNA obtained from bacteriophage Mid  part of DNA obtained from plasmid Features of phagemid It contains 2961bp It has ampicillin resistant containing gene It has 2 ori genes It has Mcs It has lacZ gene Example – most commonly used phagemids are PBluescript series Construction of PBluescript marker genes  plasmid Ori  plasmid  produce dsDNA F1/M13 phage  produce ss DNA Mcs  plasmid (21 unique restriction sites) inserted in lacZ T7 &T3 promoters  produce RNA polymerase

15. Some imp characters 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 ). Production of dsDNA When a phagemid is introduced in to a host it normally replicates and produce dsDNA E.coli pBluescript II SK (+/-) phagemids

16. Production of ssDNA However, infection of a bacterial host containing a phagemid with a 'helper' phage. e.g. VCSM13 or M13K07, provides the necessary viral components to enable single stranded DNA replication and packaging of the phagemid DNA into phage particles. These are secreted through the cell wall and released into the medium. Filamentous phage (M13)retard bacterial growth but, in contrast to lambda and T7 phage, are not generally lytic . Helper phage are usually engineered to package less efficiently than the phagemid so that the resultant phage particles contain predominantly phagemid DNA. F1 Filamentous phage infection requires the presence of a pilus so only bacterial hosts containing the F-plasmid or its derivatives can be used to generate phage particles .

17. Production of 2 diff series (depend on sense + & anti sense – strand) The pBluescript II SK and KS vector series represent two orientations of the MCS within the lacZ gene encoding the N-terminal fragment of beta- galactosidase (KS represents the orientation of the MCS in which lacZ transcription proceeds from KpnI to SacI , while SK - from SacI to KpnI ). Production of RNA transcripts In vitro transcription of T3 and T7 RNA polymerase pBluescript II SK (+/-) phagemids

18. Screening of recombinants Interpretation The recombinants are screened by growing the cells in a medium containing IPTG( iso propyl β D1 thio galactopyranoside )and x-gal When a foreign gene is inserted in the LacZ the E.coli cells fails to produce β galactosidase so no hydrolysis of x-gal so the bacterial colony remains white LacZ gene produces β galactosidase it hydrolysis the x-gal and IPTG containing medium and produce blue colour White  recombinants Blue  Non recombinants

19. Uses pBluescript II phagemids are designed for DNA cloning, dideoxy DNA sequencing, in vitro mutagenesis and in vitro transcription in a single system. Advantages It can be used to provide single strand or double stranded material with out recloning Carrying capacity is higher than phage vectors Higher efficiency in transformation than phage vectors Phagemids are generally more stable than the recombinant phage vectors Screening and storage is easier

20. cosmid

21. These are artificially constructed (hybrid) Cosmids are the vectors that are made up of a plasmid and phage DNA Nomenclature of cosmid Cos  part of DNA containing cohesive sites Mid  part of DNA obtained from plasmid Construction Cosmids were first developed by collins and Hohn in 1978 Features of cosmid It can carry about 44 kbp It has antibiotic resistant containing gene (selectable marker) It has ori gene It has Mcs (poly linker) One or two cos sites (require for packaging in to phage particle) The cos sites allow the vector to be packaged and transmitted to bacteria like a phage vector

22. Cloning Steps :- Preparation . - Target DNA: the genomic DNA to be cloned has to be cut into the appropriate size range of restriction fragments , By using Restriction Enzymes , And Cut the Cosmid Cloning site ( poly linker ) by the same Restriction enzyme , Then Ligation of physically unlinked fragments.it results in the formation of long concatamers and these are used efficiently for packing Packaging - the total DNA is transferred into an appropriate E. coli host via a technique called in vitro packaging (recombinant plasmids contained in phage shells ) . - they can also be packaged in phage capsids , which allows the foreign genes to be transferred into or between cells by transduction (a process resembling viral infection )

23. In general when a phage infects a bacteria it infects with high efficiency and when it enters it gets circularized due to the presence of cos sites and undergo recombination with the bacterial genome with site directed integrated manner So during cloning of cosmid vector when the difference between cos sites is about at a distance of 50kb away from each other they could be efficiently packaged in to the head if the distance is less than <50kb or <30kbp then no packaging in to the head takes place

24. More distance between cos sites Less distance between cos sites Transduction : The phage particle with cosmid vector is now inserted in to the host bacteria that process similar to the infection of viral particle and when the DNA enters in to the host it gets circularized

25.   Plating & Growing & Selection & Isolation - Colonies are formed in selective media , just as with a plasmid vector . - The media is : Liquid media . - So that the transformed cells can be identified by plating on a medium containing the antibiotic ( Ampicillin Agar plate ). - Those cells which did not take up the cosmid would be unable to grow. Uses of Cosmid Vectors: Cosmids are used for construction of genomic libraries of eukaryotes since these can be used for cloning large fragments of DNA .

26. Advantages of cosmids Can be used as cloning vectors Cosmids are high insert capacity vectors Cosmids are used for the analysis of complex genomes Large genes can be studied intact and genetic linkage studies can be carried out at molecular level Background molecules which do not have the insert DNA or have the smaller inserts are eliminated during packaging Transformation efficiency of cosmids is high when compared to plasmids Easy screening method is found Disadvantages of cosmids Not easy to handle these are large plasmids Not stable can acquire mutations Examples of cosmid vectors Commonly used vectors include " SuperCos 1"