1.Microbial Biotechnology AND GENETIC ENGINEERING PANKAJ BHATT RESEARCH SCHOLAR MICROBIOLOGY

2. Vector Systems A vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell An ideal Vector should possess: An origin of replication (ori) A selectable marker gene Unique restriction site(s)/MCS Small size Expression control elements 3. Cloning & Expression vectors Cloning vectors are meant typically to isolate or multiply the insert in the target cell.Expression vectors specifically are for the expression of the transgene in the target cell Generally have a promoter sequence that drives expression of the transgene.Shuttle Vectors Vector containing two origin of replication or a broad host-range ori Multiplication in two different hosts possible 4. Plasmids: History, Uses & Types 5. Plasmids: basic biology Circular extrachromosomal DNA molecules found in most of the prokaryotes; and also in some yeasts, but not in higher eukaryotes (Helsinki, 1979) Provide certain additional/advantageous properties to the bacterial host viz. antibiotic resistance, degradation of complex organic compounds , production of toxins etc. Not actually the part of bacterial genome as these can mobilize from one host to another; may sometimes be present or absent from the cells of a particular host species. Therefore, nonessential for normal bacterial growth and division Size: vary from 1-200 Kb Shape: majority are covalently closed circular DNA molecules; linear plasmids- Borellia, Streptomyces sp. 6. Based on the intrinsic properties the plasmids can be: A. Conjugative and non-conjugative Conjugation is mediated by two sets of plasmid encoded genes Transfer genes (tra) & mobilising factors (mob) Tra comprise a set of atleast 12 different genes which are responsible for synthesis of pili & other surface components allowing physical contact Mob defined by atleast two regions; one coding for a mobilising protein which binds to other mob region (nic/bom) present on the plasmid DNA Conjugative plasmids have functional transfer and mobility regions (tra+, mob+) Mediate their own transfer Present in many Gram ve and some Gram +ve bacteria Are large with stringent control of DNA replication Are present in low copy number E.g. F plasmids Non-conjugative Plasmids That which lack the transfer function (tra-, mob+) Can be mobilised if other conjugative plasmid present in the same cell E.g. ColE1, CloDF13 can be mobilised by F factor 7. Interestingly, plasmids with (tra-, mob-) phenotype are neither conjugative nor can they be mobilised. Yet, if only Mob-protein coding region is lost, such plasmids can still be mobilised by availability of a functional Mob-protein supplied by another plasmid; which can act in trans on the Mob- plasmid. B. Integrative (Those which integrate with the bacterial chromosome; also called as Episome) and Non-Integrative Plasmids (do not integrate) Based on the functional properties: F Plasmids R plasmids Col Plasmids Degradative Plasmids Virulence plasmids 8. Plasmid replication The replication strategy that a plasmid uses directly affects its copy number, host range, and incompatibility group. relies on the normal host DNA replication machinery. Plasmids will carry distinct origins of replication (ori) and genes that enable and control the frequency of their replication. Theta mode of replication Most of the Gram ve bacteria e.g. ColE1, RK2 Rolling circle mode of replication Some Gram +ve bacteria 9. Plasmid Incompatibility Two plasmids are incompatible if they are unable to coexist in the same cell Usually these share a common replication system and common types of pilli, thus compete with each other during replication Based on this, more than 30 incompatibility groups (Inc) have been defined in E. coli and 13 in S. aureus. 10. Plasmid Host Range- determined by its ori region Narrow host range plasmids: most of the naturally occurring plasmids have a restricted host range. E.g. ColE1 and its derivatives grow only in E. coli and related organisms such as Salmonella. Broad host range plasmids: Possess less specific ori region such that it can function in a number of bacterial species. Most of them require very few of the host-encoded proteins Generally plasmids of the incompatibility group P, Q, and W have a broad host range; also called as promiscuous plasmids e.g. RP4, RSF1010, RK2 etc. Can replicate in a no. of bacteria such as E. coli, Agrobacterium, Pseudomonas, Rhizobium etc. Thus interesting for gene manipulation experiments!!! 11. Plasmid copy number: the no. of molecules of an individual plasmid that may normally be found in the single bacteria are referred to as the Copy Number Low copy no. (STRINGENT PLASMIDS) Copy no. hardly ever >1 Rigid control of replication Copy no. either controlled by or correlated with the replication of chromosomal DNA molecule Thus, pDNA replicates only once or twice before the cell division Single copy plasmids have a partitioning system; so as to ensure segregation of duplicated copies to different daughter cells High Mol. Wt. & ConjugativeHigh copy no. (RELAXED PLASMIDS) multiple copies per cell Relaxed control of replication pDNA replication not dependent upon host cell chromosomal DNA replication pDNA replicates repeatedly until a proper no. is reached No such partitioning system +nt Generally Low Mol. Wt and non-conjugative 12. Plasmids as Vectors: essential features Autonomous replication in host: Ori region Selectable marker(s) Unique restriction sites/Multiple cloning sites Low molecular weight High copy number Size: small 13. A more detailed look at plasmids Promotor Site Origin of ReplicationAntibiotic Resistance GeneMultiple Cloning Site 14. Cloning into a Plasmid 15. Some earlier used plasmids pSC101 the first cloning vector, used in 1973 by Boyer and Cohen. 9 kb, low copy no. the original pSC101 only contained tetracycline resistance and a restriction site for EcoRI, the commercially available pSC101 gained restriction sites for several enzymes, including HindIII, in addition to the EcoRI site. 16. ColE1 plasmid Approx 6.4 kb Exist as multiple copies per cell Single EcoR1 site present in colicin production (cea) gene; transformants selected by inactivation of colicin production; technically difficult and cell resistance to colicins arises spontaneously at quite high frequency in a bacterial population. 17. Earlier natural plasmids of E. coli were used as cloning vectors e.g. pSC101, pSF2124, ColE1 etc. Disadvantages: Generally large sizes difficult transformation Presence of non-essential genes (toxin production etc.) Absence of unique restriction sites, desired marker genes Conjugative and mobilization propertiesTherefore, artificial construction of desired plasmid vectors Advantages: Increased efficiency of transformation Easier to restriction map Higher copy numbers 18. pBR322 an example of an early cloning vector that replicates in E. coli Constructed by Bolivar & Rodriguez Approx 4.3kb plasmid composed of sequences from ColE1, pMB1 and pSC101 plasmids ori allows independent replication single cleavage sites for various restriction enzymes (BamHI etc) tet resistance gene amp resistance gene Mechanism: insertion of foreign DNA at BamHI site tet resistance gene inactivated transformants carrying foreign DNA are amp resistant but tetracycline sensitive Double selection of transformants required /replica plating 19. pUC series 1982, University of California Approx. 2.7kb in size; high copy no. amp resistance gene ori derived from pBR322 Unique restriction sites clustered into one short fragment called as the multiple cloning site (MCS) or the polylinker site 20. Another advancement was the use of blue white screeningThe Lac Z gene coding for beta-galactosidase was incorporated as a part of the MCS region in plasmids Insertional Inactivation of the lac Z gene leads to the selection of transformants 21. pGEM series pGEM3Z/ pGEM4Z next generation introduces RNA expression for in vitro or in vivo expression 22. Limitations of Cloning in Plasmids Upper limit for clone DNA size is 12 kbRequires the preparation of competent host cellsInefficient 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 23. Bacteriophage lambda () as cloning vehicles 24. Life cycle of a phage 25. Phage replication circles multiply by theta form and continues for 515 minutes after infection. Rolling circles predominates after 15 minutes and produce linear concatemers (genomes linked end to end) cos sites recognised by endonucleases/terminase producing individual phage genomes for packaging Packaging also requires THF (termination host factor) provided by the host cell. 26. Molecular biology of genome Mol wt. 31X106 Da, 48.5 Kb long DNA isolated from virus particles is a ds linear molecule cos sites: 12 nucleotides long single stranded cohesive termini at each 5 end When inside host cell: circularization of phage DNA Genetic map of : almost 40 genes organised in functional clusters5 cos sites 5 cos sitesHeadTailLeft regionRecombination, LysogenyRegulation, Replication, LysisCentral region Much of this central region is not Much of this central region is not essential for lytic growth and can essential for lytic growth and can be deleted or replaced for the be deleted or replaced for the construction of suitable vectors construction of suitable vectorsRight region 27. Lysis Replication oricosHead TailLysis Replication oriCircularized lambdaLysogenyDeletion of non essential region Deletion of non essential region makes room for the foreign makes room for the foreign insert insertcosHead Tail 28. Constraints with wild type ; construction of Vectors Protein capsule of has a tight constraint on the amount of DNA that will fit inside it Molecules