Host cell and vectors

About the host and vectors used in Biotechnology

1. Host cells and vectors

2. Host cells and vectors Gene cloning is concerned with the selection and use of a suitable carrier molecule or vector, and a living system or host in which the vector can be propagated Host Gene for structural analysis- simple system Express the genetic information - more specific system simple primary host is used to isolate a sequence that is then introduced into a more complex system for expression. A  host is an organism that harbors a  parasite, or a mutual or  commensal symbiont , typically providing nourishment and shelter

3. Host cell types Prokaryotic hosts Eukaryotic hosts Prokaryotic hosts Easy to handle and propagate Available as a wide variety of genetically defined strains Accept a range of vectors. Essential features of Host Gram-negative bacterium One of the simplest host cell Single chromosome packed into a compact structure known as the nucleoid Genome size - 4.6 × 106 base pairs E. coli

4. Processes of gene expression (transcription and translation) are coupled No post-transcriptional modification of the primary transcript Many genetically different strains available for specific applications Bacillus Pseudomonas Streptomyces O ther bacteria used as hosts Gram-positive bacteria Gram-positive bacteria are well known for their contributions to agricultural, medical and food biotechnology and for the production of recombinant proteins. Bacillus

5. Bacillus subtilis  - an attractive host because of several reasons: Non-pathogenic and is considered as a GRAS organism (generally regarded as safe)   No significant bias in codon usage Capable of secreting functional extracellular proteins directly into the culture medium (at present, about 60% of the commercially available enzymes are produced by Bacillus species) A large body of information concerning transcription, translation, protein folding and secretion mechanisms, genetic manipulation and large-scale fermentation has been acquired. Disadvantages Lacks the membrane bound nucleus (and other organelles) found in eukaryotic cells Certain eukaryotic genes may not function in E. coli May not be easy to ensure that a prokaryotic host produces a fully functional protein

6. Eukaryotic hosts Ranges from microbes ( yeast and algae) - complex multicellular organisms (ourselves Yeast - Saccharomyces cerevisiae amenable to classical genetic analysis range of mutant cell types is available 3.5 times more DNA than E. coli Fungi - Aspergillus nidulans and Neurospora crassa Algae - Chlamydomonas reinhardtii Plants - Protoplasts, Intact cells ,Whole organism Animals - Insect cells - Drosophila melanogaster Mammalian cells Oocytes Whole organism

7. vectors Essential features of Vectors DNA molecule that functions as a “molecular carrier” that carry the DNA of interest into the host cell & facilitates its replication. P ossess an origin of replication ( ori ). Be easy to isolate,  i.e . small. Be non-toxic to host cells. Have space for foreign inserts. Have unique restriction sites for common restriction enzymes. Have convenient markers for selection of transformants ,  e.g . antibiotic resistance genes. Relaxed,  i.e . multiple copies in a host cell.

8. Most prokaryotic vectors are based on 1.   Plasmids 2.  Bacteriophages 3.  Cosmids   Plasmids Extra chromosomal genetic elements Not essential for bacteria to survive Confer advantageous traits (such as antibiotic resistance ) on the host cell Cleaved by restriction enzymes, leaving sticky or blunt ends. Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid. What are plasmids?

9. Plasmid classification The most useful classification of naturally occurring plasmids is based on the main characteristic coded by the plasmid genes Fertility plasmid carry only tra genes and have no characteristic beyond the ability to promote conjugal transfer of plasmids example : F plasmid of E. coli. (2) Resistance or R plasmids carry genes conferring on the host bacterium resistance to one or more antibacterial agents, such as chloramphenicol,ampicillin and mercury Important in clinical microbiology-treatment of bacterial infections example : RP4,found in Pseudomonas, also occurs in many other bacteria (3) Col plasmids code for colicins proteins that kill other bacteria example : ColEl of E. coli. (4) Degradative plasmids allow the host bacterium to metabolize unusual molecules such as toluene and salicylic acid example : TOL of Pseudomonas putida . (5) Virulence plasmids confer pathogenicity on the host bacterium example : Ti plasmids of Agrobacterium tumefaciens , which induce crown gall disease on dicotyledonous plants.

10. conjugative and non-conjugative plasmids Conjugative plasmids -conjugation process which requires functions specified by the tra (transfer) and mob ( mobilising ) regions Non-conjugative plasmids - not selftransmissible but may be mobilised by a conjugation-proficient plasmid if their mob region is functional Low-copy-number and Highcopy - number plasmids Low-copy-number plasmids - exhibit stringent control of DNA replication replication of the pDNA depend on host cell chromosomal DNA replication. Highcopy - number plasmids - relaxed plasmids not dependent on host cell chromosomal DNA replication. Other classification of plasmids The copy number refers to the number of molecules of an individual plasmid that are normally found in a single bacterial cell Conjugative plasmids -large, stringent control, low copy numbers Nonconjugative plasmids -small, relaxed DNA replication, high copy numbers

11. Properties of some naturally occurring plasmids Ap , ampicillin ; Cm, chloramphenicol ; Km, kanamycin ; Sm , streptomycin; Sn , sulphonamide ; Tc , tetracycline. E1imm and DF13imm represent immunity to the homologous but not to the heterologous colicin Basic cloning plasmids In naming plasmids, p is used to designate plasmid, and this is usually followed by the initials of the worker(s) who isolated or constructed the plasmid. Numbers may be used to classify the particular isolate.

12. pBR322 developed by Francisco Bolivar and his colleagues (The p stands for "plasmid," and BR for " Bolivar" and "Rodriguez.“ ) Construction of pBR322 involved a series of manipulations -DNA from three sources (the replicon of plasmid pMB1, amp R  gene -RSF2124, tet R  gene- pSC101) features of this plasmid low molecular weight, antibiotic resistance genes, an origin of replication, and several single-cut restriction endonuclease recognition sites Amp - ampicillin Tet - tetracycline and ori - origin of replication Map of plasmid pBR322.

13. pUC18 - plasmid cloning vectors  Vieira and Messing in 1982 UC represents-   University of California N-terminal fragment - lac Z gene Insertional inactivation method ( Blue – white selection) pUC18  

14. Some commercially available plasmid vectors

15. What are bacteriophages ? Bacteriophages 1940s Max Delbruck ‘eaters of bacteria’ - viruses that are dependent on bacteria for their propagation. phages fall into three main groups: (1) tailless, (2) head with tail and (3) filamentous genetic material-single or double-stranded DNA or RNA In tailless and tailed phages the genome encapsulated in an icosahedral protein shell called a capsid (sometimes known as a phage coat or head). Typical dsDNA phages- genome makes up about 50% of the mass of the phage particle phages -relatively simple systems when compared to bacteria-used as models for the study of gene expression

16. temperate ( lysogenic life cycles ) Classification of phages Depending on life cycle virulent ( lytic life cycle ) Bacteriophage lambda as a vector First viral cloning vector in 1974. Preparing genomic libraries Hold a larger piece of DNA than a plasmid vector G enome - 48.5 kb in length encodes 46 genes At the ends of the linear genome there are short (12 bp ) single-stranded regions that are complementary. These act as cohesive or ‘sticky’ ends, which enable circularization of the genome The region of the genome that is generated by the association of the cohesive ends is known as the cos site Insertion vectors have unique restriction endonuclease sites that allow the cloning of small DNA fragments in addition to the phage λ genome.

17. Used for preparing cDNA expression libraries. The recombinant viral particle infects bacterial host cells, in a process called “ transduction” The host cells lyse after phage reproduction, releasing progeny virus particles. The viral particles appear as a clear spot of lysed bacteria or “ plaque” on an agar plate containing a lawn of bacteria. Each plaque represents progeny of a single recombinant phage and contains millions of recombinant phage particles. Most contemporary vectors carry a lacZ ′ gene allowing blue-white selection.

18. M13 phage M13 -.   structural elements: Circular, single-stranded DNA ~6.4 kb long 10 genes in the genome. filamentous phage, infect  E. coli  through pili , able to produce new virions without lysing the host cell Gene II: codes for nickase , allows rolling-circle replication Gene III: codes for the pilot protein, which guides the nascent ssDNA to the membrane, Gene VIII: coat protein, encapsulates the pilot protein and the ssDNA phage DNA as it extrudes through the membrane

19. Cosmids A hybrid vector made up of plasmid sequences and the cohesive ends ( cos sites) of bacteriophage lambda. Cosmids can be packaged in lambda phage particles for infection into E. coli. This permits cloning of larger DNA fragments (up to 45 kb) Cosmids and cosmid recombinants replicate as plasmids. Likely to be less stable than plasmids because of large insert and high copy number .

20. Phagemids contain the f1 (M13) phage origin of replication developed to overcome the size limitation of the M13 cloning system applications DNA sequencing and the production of probes for hybridization . Example pEMBL9 or pBluescript 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.

21. Why do we need artificial chromosomes? T ransformation of novel genes into plants and animals has employed various methods for delivering DNA into cells ( eg . transfection , microinjection,  Agrobacterium  infection) T hese methods all ultimately depend upon the DNA repair mechanisms of the target cell to insert the DNA into chromosomes at a random location, anywhere in the genome. Advantages of artificial chromosomes Random insertion can potentially disrupt important genes. Artificial chromosomes don't require insertion of sequences into naturally-occurring chromosomes. N o limit to the number of genes or size of fragments that could be inserted using artificial chromosomes. Because all inserted genes on an artificial chromosome are all linked, they will not segregate independently, making it easy to cross the transformed genes into a new genetic background. easily control copy number with artificial chromosomes . With random insertion, the level of expression is highly dependent on the site of insertion. Insertion near a strong enhancer could cause very strong expression Artificial chromosomes

22. new generation vectors clone large pieces of DNA fragments up to 100 – 750 kb YACs are designed to replicate as plasmids in bacteria when no foreign DNA is present. Once a fragment is inserted, YACs are transferred to cells, they then replicate as eukaryotic chromosomes. Linear DNA vectors Telomers - Stabilize chromosome ends Centromer -ensures chromosome partitioning between two daughter cells A and B: selectable markers YAC can use both yeast and bacteria as a host Yeast Artificial Chromosomes (YACs)

23. BACs can hold up to 300 kbs . The F factor of E.coli is capable of handling large segments of DNA. Recombinant BACs are introduced into E.col i by electroporation ( a brief high-voltage current). Once in the cell, the rBAC replicates like an F factor. Example: pBAC108L Regulatory genes- OriS and repE - control F-factor replication parA and parB - limit the number of copies to one or two. A chloramphenicol resistance gene Bacterial Artificial Chromosomes(BACs )

24. P1-derived artificial chromosomes (PACs) Developed by Ioannou et al Incorporates the features of phage P1 and F-factor systems Transformed into the E. coli host by electroporation Insert range 100-300 kb No major problems with chimaerism or clone instability

25. Mammalian artificial chromosomes (MACs) Main features for an efficient mammalian AC (MAC) a vectorial capacity up to a few megabase (2) a manageable size for their in vitro manipulation (3) a correct intracellular location and copy number (4) no untoward effect on the host cell (5) The ability to express the transgene (or transchromosome ) in a physiological way A pre-engineered platform MAC with multiple acceptor sites [Artificial Chromosome Expression System (ACE system) ] Capable of harboring a number of different genes Large carrying capacity Represents a non-integrating safe vector. ACE Integrase , a lambda integrase enzyme, which has been modified to render the integrase functionally independent of bacterial host cell factors and capable of operating in a mammalian context. ACE system)

26. ACE targeting vector (ATV) is a plasmid-based shuttle vector that conveys a gene(s) of interest onto Platform ACE by means of targeted recombination between the recombination acceptor attP sites present on Platform ACE and the recombination donor attB site of the ATV, catalyzed by the ACE Integrase . The ACE system is a platform technology for protein production, transgenesis and gene therapy.

27. microchromosome can act as a new chromosome in a population of human cells. That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very small roughly 6-10  megabases in size able to carry new genes introduced by human researchers. Appeared in 1997 (Willard) useful in expression studies as gene transfer vectors and are a tool for elucidating human chromosome function. Grown in HT1080 cells, they are  mitotically and cytogenetically stable for up to six months Human artificial chromosome (HAC)

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