DESIGN OF PLASMID VECTOR & PROKARYOTIC AND EUKARYOTIC VECTORS

The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952

1. DESIGN OF PLASMID VECTOR & PROKARYOTIC AND EUKARYOTIC VECTORS SUBMITTED BY ; GEDIYA LALJI N REG. NO. : 04-2854-2016

2. DESIGN OF PLASMID VECTORS

3. Joshua Lederberg The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952 Joshua Lederberg was an American molecular biologist known for his work in genetics, artificial intelligence, and space exploration. He was just 33 years old when he won the 1958 Nobel Prize in Physiology or Medicine for discovering that bacteria can mate and exchange genes. He shared the prize with Edward L. Tatum and George Beadle who won for their work with genetics.

4. What is a plasmids ? Plasmids are double stranded, closed circular covalently bonded DNA molecules, which exist in the cell as extra chromosomal units. They are self-replicating and found in a variety of bacterial species. e.g. E. coli, Shijella , Erwinia , Streptococcus, Vibrio, Salmonella, Agrobacterium, Enterobactor , Rhizobium , etc. British Genetist Joshua Lederberg (1952) first use this term. Plasmid Host Cell P ACYC177 Escherichia Coli P BR322 Escherichia Coli P BR324 Escherichia Coli P MB9 Escherichia Coli P RK646 Escherichia Coli P C194 Staphylococcus aereus P SA0501 Staphylococcus aereus P BS161-1 Basillus Subtilis P WWO Pseudomonas Putida

5. Features of plasmid Plasmids are most often found in cytoplasm of bacterial cell. It is a circular, double-stranded DNA molecule. Most of the plasmids are small in size and contain a few numbers of genes. Plasmids genes are replicated, Transcripted and translated with a chromosomal gene independently. The atomic weight of plasmid is 1×10 6 to 200×10 6 Plasmid has a 200-10000 bp and contains1-50 genes. It easily separates from the bacterial chromosome. It may be transferred from one cell to other cell and also one species to other species of bacteria. As like as nuclear DNA, plasmids are attached with histon . It has a replication ori . Anti-resistance genes of plasmids are used as a selectable marker. Replication system of plasmid is unidirectional or bidirectional. They exists separately from main chromosome. Some plasmids can integrate with chromosome and exists along with it. This type of plasmids are called episomes . For example F factor of E.coli is an episome .

6. What types of plasmids are there? Types of plasmids by their ability to transfer to other bacteria – three types Conjugative plasmids: Able to perform conjugation, which is the process of transferring plasmids to another environment Non-conjugative plasmids: Unable to perform conjugation Intermediate plasmids: Able to be transferred by conjugative plasmids. They contain a subset of genes

7. Types of plasmids by their function- five types Fertility Plasmids Contain transfer genes and are capable of conjugation Resistance Plasmids Contain genes that can build resistance to antibiotics or poisons and help bacteria produce pili . Col Plasmids Contain genes that code for proteins that kill other bacteria Degradative Plasmids Allows to digest unusual substances Virulence Plasmids Turn bacterium into a pathogen

8. Shapes of plasmids There are 5 shapes of plasmids Nicked Open-Circular: DNA has one strand cut Relaxed Circular: DNA is fully intact Linear: DNA has free ends Supercoiled: DNA is fully intact but has a twist in it, making it more compact Supercoiled Denatured: Slightly less compact than supercoiled

9. Plasmid parts A vector is a DNA molecule used to carry genes from organism to organism . Plasmids have 3 key parts. They have an origin of replication, a selectable marker gene, and a cloning site. The origin of replication is used to indicate where DNA replication is to begin. The selectable marker gene is used to distinguish cells containing the plasmid from cells that don’t contain it. The cloning site is a site in the plasmid where the DNA is inserted.

10. Plasmids are first type of cloning vector developed.These are used as vector to clone DNA in bacteria. Engineered plasmids can be used to clone genetic material of up to 10,000 base pairs. Examples : pBR322 plasmid pUC18 plasmid Plasmid – a vector

11. Ti Plasmid The most commonly used vector for gene transfer in higher plants are based on tumour inducing mechanism of the soil bacterium A. tumefaciens , which is the causal organism for crown gall disease. The disease is caused due to transfer of DNA segment from the bacterium to the plant nuclear genome. The DNA segment which is transferred is called T-DNA and is part of a large Ti plasmid found in virulent strains of A. tumefaciens . Most Ti plasmids have four regions in common Region A: comprising T-DNA which is responsible for tumour induction leading to production of tumors with altered morphology ( shooty or rooty mutant galls). Sequences homologous to this region are always transferred to plant nuclear genome, so that the region is described as T-DNA (transferred DNA) Region B: This region is responsible for replication Region C Responsible for conjugation Region D: Responsible for virulence, so that mutation in this region abolishes virulence. This region is therefore called virulence ( vir ) region and plays a crucial role in the transfer of T-DNA into the plant nuclear genome.

12. The T-DNA consist of following regions: An ONC region: Consisting of 3genes (2 genes tms1 and tms2 rep shooty locus and one gene tmr rep rooty locus) responsible for 2 phytohormones , namely IAA and isopentyladenosine-5’-monophosphate (a cytokinin ). These genes encode the enzymes responsible for the synthesis of these phytohormones so that the incorporation of these genes in plant nuclear genome leads to the synthesis of phytohormones in the host plant. These phytohormones in turn alter the development programme , leading to the formation of crown gall. An OS region: responsible for synthesis of opines. These are derivatives of various compounds ( eg . Arginine + pyruvate) that are found in plant cells. Two most common opines are octopine and nopaline . The enzymes for sytnhesis of these 2 opines, are encoded by ( octopine synthase and nopaline synthase) T-DNA. Depending upon the opine it codes, they are described as octopine type Ti plasmid or nopaline type Ti plasmid.

13. The T-DNA region in the plasmid is flanked on both the sides with 25 bp direct repeat sequences, which are essential for T-DNA transfer, acting only in its cis orientation. Any DNA sequences flanked by these 25 bp repeated sequence in the correct orientation, can be transferred to plant cells. This is successfully utilized for Agrobacterium mediated gene transfer in higher plants.

14. Plasmids in genetic engineering In genetic engineering, plasmids provides a versatile tool that are used to make copies of particular genes. This is done by inserting the gene to be replicated into the plasmid, then inserting the plasmids into bacteria by a “Transformation”.

15. One more key use of plasmids is to make large amounts of proteins.

16. Various applications

17. PROKARYOTIC AND EUKARYOTIC VECTOR

18. CHARACTERISTICS Self replication, multiple copies. Replication origin site. Cloning site. Selectable marker gene. Small size. Low molecular weight. Easily isolated & purified. Easily introduced into host cell. Control elements – promoter, operator, ribosome binding site . Restriction sites. VECTORS Small DNA molecule capable of self replication . Cloning vehicle or Cloning DNA . Carrier of DNA fragment . E.g. Plasmid, Phage, Hybrid vector, Artificial Chromosomes.

19. TYPES Two types :- Cloning Vectors Propagation or cloning of DNA insert inside a suitable host cells. Examples: Plasmids, Phage or Virus Obtaining millions of copies. Uses :- Genomic library. Preparing probes. Genetic Engineering Experiments. Selection of cloning vector depends on :- (a) Objective of cloning experiment (b) Ease of working. (c) Knowledge existing about the vector. (d) Suitability. (e) Reliability.

20. Expression Vectors Express the DNA insert producing specific protein. They have prokaryotic promoter. Ribosome binding site. Origin of replication. Antibiotic resistance gene. Expression vectors with strong promoters. Inducible Expression Vectors. Eukaryotic expression vectors.

21. Six different types of cloning vectors : Plasmid cloning vector Phage  cloning vector Cosmid cloning vector Shuttle vectors Yeast artificial chromosome (YAC) Bacterial artificial chromosome (BAC) Fosmid cloning vector

22. Plasmid Cloning Vectors: Bacterial plasmids , naturally occurring small ‘ satellite ’ chromosome, circular double-stranded extrachromosomal DNA elements capable of replicating autonomously. Plasmid vectors engineered from bacterial plasmids for use in cloning. Feeatures (e.g., E. coli plasmid vectors): Origin sequence ( ori ) required for replication. Selectable trait that enables E. coli that carry the plasmid to be separated from E. coli that do not (e.g., antibiotic resistance, grow cells on antibiotic; only those cells with the anti-biotic resistance grow in colony). Unique restriction site such that an enzyme cuts the plasmid DNA in only one place. A fragment of DNA cut with the same enzyme can then be inserted into the plasmid restriction site. Simple marker that allows you to distinguish plasmids that contain inserts from those that do not (e.g., lacZ + gene )

23. EXAMPLES OF PLASMID VECTORS pBR 322 pBR 327 pBR 325 pBR 328 pUC 8 pUC 19 pUC 12 pUC 13 pGEM 3 Z Plasmid

24. pUC19 Polylinker : restriction sites Origin sequence Ampicillin resistance gene lacZ + gene

25. Detailed map showing polylinker region in pUC57 (genecript.com)

26. *Cut with same restriction enzyme *DNA ligase

27. Some features of pUC19 plasmid vector: High copy number in E. coli , ~100 copies/cell, provides high yield. Selectable marker is amp R . Ampicillin in growth medium prevents growth of all other E. coli that do not contain plasmid. Cluster of several different restriction sites called a polylinker occurs in the lacZ ( - galactosidase ) gene . Cloned DNA disrupts reading frame and - galactosidase production. Add X-gal to medium; turns blue in presence of - galactosidase . Plaque growth : blue = no inserted DNA and white = inserted DNA . Finally, plasmids are transformed into E. coli by chemical incubation or electroporation (electrical shock disrupts the cell membrane). Good for <10kb; Cloned inserts >10 kb typically are unstable.

28. PBR 322 Cloning Vector. 15 copies. Reconstructed plasmid. Derived from Ecoli plasmid- ColE1 . P B R 322 – 4362 base pairs P – denotes Plasmid B – Scientific Boliver R - Rodriguez 322 – number given to distinguish. Ampilicin resistance gene derived from RSF2124. Tetracycline resistance gene from PSC101. Origin of replication from PMB1. Two selectable markers amp r tet r

29. Plasmid Vector: pBR322 Contains: Selectable Markers : Ampicillin resistance gene. Tetracycline resistance gene. Col E I replication origin. Eco RI site. Structure of E.Coli plasmid cloning vector pBR322

30. Bacterial Vectors Continued… Cloning large DNA fragmance . Linear Phage molecule. Efficient than plasmid . Used in storage of recombinant DNA. Commonly used Ecoli phages :- λ phage M13 Phage PHAGE

31. Bacteriophage: A virus that infects bacteria, and whose DNA can be engineered into a cloning vector. Kills bacteria by two pathways: Lytic pathway Bacteriophage DNA is inserted into the bacteria, and phage DNA and phage proteins are made, assembled, and burst out of the bacteria, causing the bacteria to burst (called “ lysis ”). Important in research because recombinant phage DNA can be inserted into bacteria on a bacterial plate. The bacteria die, forming clear areas called “plaques” where the recombinant DNA can be isolated. Lysogenic pathway: The bacteriophage genome is integrated into the bacterial DNA, replicating along with the bacterial cell genome.

32. Phage  cloning vectors: Engineered version of bacteriophage  (infects E. coli ). Central region of the  chromosome (linear) is cut with a restriction enzyme and digested DNA is inserted. DNA is packaged in phage heads to form virus particles. Phages with both ends of the  chormosome and a 37-52 kb insert replicate by infecting E. coli . Phages replicate using E. coli and the lytic cycle (see Fig .). Produces large quantities of 37-52 kb cloned DNA. Like plasmid vectors, large number of restriction sites available; phage  cloning vectors useful for larger DNA fragments than pUC19 plasmid vectors.

33. Phage M 13 Vectors Filamentous bacteriophage of Ecoli . Used for obtaining single stranded copies. DNA sequencing. Single stranded. Inside host cell become double stranded.

34. HYBRID VECTOR Component from both plasmid & phage chromosomes. Helper phage provided. Developed in 1978 by Barbara Hohn & John Collins. 30 – 40 Kb Origin of replication, cloning site, marker gene, DNA cos site. Smaller than plasmid. Use – construction of genomic libraries of eukaryotes. e.g. Cosmid

35. Cosmid cloning vectors: Features of both plasmid and phage cloning vectors . Do not occur naturally; circular . Origin ( ori ) sequence for E. coli . Selectable marker, e.g. amp R . Restriction sites . Phage  cos site permits packaging into  phages and introduction to E. coli cells . Useful for 37-52 kb.

36. Cloning by using Cosmid vectors

37. Shuttle vectors: Capable of replicating in two or more types of hosts. Replicate autonomously, or integrate into the host genome and replicate when the host replicates. Commonly used for transporting genes from one organism to another (i.e., transforming animal and plant cells). Example: *Insert firefly luciferase gene into plasmid and transform Agrobacterium . *Grow Agrobacterium in large quantities and infect tobacco plant.

38. ARTIFICIAL CHROMOSOME Linear or Circular. 1 0r 2 copies per cell . Different types – Bacterial Artificial Chromosome (BAC) Yeast Artificial Chromosome (YAC) P1 derived artificial chromosome (PAC) Mammalian Artificial Chromosome (MAC) Human Artificial Chromosome . (HAC) YAC – Cloning in yeast BAC & PAC – Bacteria MAC & HAC – Mammalian & Human cells.

39. Bacterial Artificial Chromosomes (BACs) : Vectors that enable artificial chromosomes to be created and cloned into E. coli . Features : Useful for cloning up to 200 kb, but can be handled like regular bacterial plasmid vectors. Useful for sequencing large stretches of chromosomal DNA; frequently used in genome sequencing projects. Like other vectors, BACs contain: Origin ( ori ) sequence derived from an E. coli plasmid called the F factor. Multiple cloning sites (restriction sites). Selectable markers (antibiotic resistance).

40. Yeast Artificial Chromosomes (YACs) : Vectors that enable artificial chromosomes to be created and cloned into yeast. Features : Yeast telomere at each end. Yeast centromere sequence. Selectable marker (amino acid dependence, etc.) on each arm. Autonomously replicating sequence (ARS) for replication. Restriction sites (for DNA ligation). Useful for cloning very large DNA fragments up to 500 kb; useful for very large DNA fragments.

41. Fosmid : Based on the E. coli bacterial F-plasmid. Can insert 40 kb fragment of DNA. Low copy number in the host (e.g., 1 fosmid ). Fosmids offer higher stability than comparable high copy number cosmids . Contain other features similar to plasmids/ cosmids such as origin sequence and polylinker .

42. What determines the choice vector? insert size vector size restriction sites copy number cloning efficiency ability to screen for inserts what down-stream experiments do you plan? Table

43. Plant Cloning Vectors Used for purposes such as resistance to disease, pests, and herbicides; improving crop quality and yield; improving nutritional quality; and increasing the shelf life of foods. Most commonly used plant vectors are the tobacco mosaic virus and the Ti plasmid from the soil bacterium Agrobacterium tumeifaciens . About Agrobacterium tumeifcaciens and the Ti plasmid: Agrobacterium tumeifcaciens causes crown gall disease (tumor formation) in plants, caused by T-DNA (transferred DNA), located in the Ti plasmid, and contains eight genes that integrate into the plant genome. Engineered Ti plasmids lack the tumor-causing genes, but have the genes required to integrate the DNA of interest into the plant genome The plasmid is inserted into a plant embryo either by soaking seeds with recombinant A. tumeifaciens bacteria, or by inserting the Ti plasmid into cells, which will give rise to the entire plant. Selectable marker genes will allow for the selection of only the plant cells with the plasmid DNA .

44. Mammalian Cell Vectors There are several mammalian cell vectors: Simian virus 40 (SV40)- a small DNA tumor virus, could only hold a small piece of DNA and caused only transient (temporary) expression of the inserted DNA. Retrovirus- a single-stranded RNA virus that contains a gene for the enzyme reverse transcriptase to create double-stranded DNA from RNA template, so that the DNA can integrate into the host cell’s genome. It needs to infect actively dividing cells. Adenovirus- a double-stranded DNA virus that can infect many types of host cells with high efficiency, with a low chance for causing disease. It does not have to infect actively dividing cells. Mammalian cells are used because bacteria are not able to produce complex eukaryotic proteins that are modified by processes such as glycosylation, or if the mRNA needs to be processed after transcription.