Types of vectors, Prokaryotic and Eukaryotic

vectors types
prokaryotic and eukaryotic
prokaryotic vectors= 1. ti-plasmid 2. pUC18 3. pBR322 4. lambda phage
Eukaryotic vectors= 1.BAC 2. YAC

by
Malaka Madhusudhana Reddy
Research Scholar
Dept.of :: Zoology
Yogivemana University, kadapa.
Andhrapradesh, India.

1. MALAKA MADHUSUDHANA REDDY Msc Research scholar Dept.of :: Zoology Yogivemana university, K adapa

2. What is the difference between cloning and expression vectors? Cloning vectors are used to increase the number of copies of the cloned gene or to amplify a foreign gene. Expression vectors are used to increase the expression of the foreign gene product. Cloning  is the process of producing  genetically  identical individuals of an  organism  either naturally or artificially. In nature, many organisms produce clones through  asexual reproduction . Cloning in  biotechnology  refers to the process of creating clones of organisms or copies of  cells  or  DNA  fragments ( molecular cloning ). Beyond   biology , the term refers to the production of multiple copies of  digital media  or  software . Cloning

3. Cloning Vector A  cloning vector  is a small piece of  DNA , taken from a  virus , a  plasmid , or the  cell  of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for  cloning   purposes. The   vector  therefore contains features that allow for the convenient insertion or removal of a DNA fragment to or from vector, for example by treating the vector and the foreign DNA with a  restriction enzyme  that cuts the DNA. DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined together by  molecular ligation . After a DNA fragment has been cloned into a cloning vector, it may be further  subcloned  into another vector designed for more specific use .

4. A cloning vector is a DNA molecule in which foreign DNA can be inserted or integrated and which is further capable of replicating within host cell to produce multiple clones of recombinant DNA. Such a vector is called Cloning Vector. Plasmids and phages are the vectors used for cloning purposes, particularly in prokaryotes (bacteria ). Origin of replication ( ORI) This process marks autonomous replication in vector. ORI is a specific sequence of nucleotide in DNA from where replication starts. When foreign DNA is linked to this sequence then along with vector replication, foreign (desirable) DNA also starts replicating within host cell. Selectable Marker Besides ORI, a cloning vector should have selectable marker gene. This gene permits the selection of host cells which bear recombinant DNA (called transformants ) from those which do not bear rDNA (non- transformants ). Restriction sites It should have restriction sites, to allow cleavage of specific sequence by specific Restriction Endonuclease. Restriction sites in E.coli cloning vector pBR322 include HindIII , EcoRI , BamHI , SalI , PvuI , PstI , ClaI   etc.

5. Figure 20.3-1 Restriction enzyme cuts sugar-phosphate backbones. Restriction site DNA 5  5  5  5  5  5  3  3  3  3  3  3  1 Sticky end GAATTC CTTAAG CTTAA G AATTC G

6. Figure 20.3-2 One possible combination DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. Restriction enzyme cuts sugar-phosphate backbones. Restriction site DNA 5  5  5  5  5  5  5  5  5  5  5  5  5  5  3  3  3  3  3  3  3  3  3  3  3  3  3  3  2 1 Sticky end GAATTC CTTAAG CTTAA G AATTC G G G AATTC CTTAA G G G G AATT C AATT C C TTAA C TTAA

7. Figure 20.3-3 Recombinant DNA molecule One possible combination DNA ligase seals strands DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. Restriction enzyme cuts sugar-phosphate backbones. Restriction site DNA 5  5  5  5  5  5  5  5  5  5  5  5  5  5  5  5  3  3  3  3  3  3  3  3  3  3  3  3  3  3  3  3  2 3 1 Sticky end GAATTC CTTAAG CTTAA G AATTC G G G AATTC CTTAA G G G G AATT C AATT C C TTAA C TTAA

8. cloning vector Prokaryotic vectors pUC18 pBR322 Lambda phase Ti-plasmid E ukaryotic vectors BAC YAC

9. Cloning Vector pUC18

10. Cloning Vector pUC18 pUC18 high copy cloning vector for replication in E. coli, suitable for “blue-white screening” technique. pUC18 is a small, high copy cloning vector for replication in E. coli. It has been constructed using the ampicillin resistance gene and the pMB1 origin of replication from pBR322. It is a circular double stranded DNA and has 2686 base pairs. The pMB1 of pUC18 differs from the pBR322 origin by a single point mutation and the lack of the rop gene  (also known as  repressor of primer ) , leading to a high copynumber .   A  plasmid  is a  circular dsDNA  molecule  a few hundred or thousand base pairs in circumference. The artificial plasmid  pUC18  has been genetically engineered to include ( 1 ) a gene for  antibiotic resistance  to  Ampicillin  ( amp R ), and ( 2 )  a gene (and its  promoter ) for the enzyme  beta- galactosidase   ( lacZ ). The  lacZ  gene contains a ( 3 )  polylinker  region, with a series of unique restriction sites found nowhere else in the plasmid. Digestion with any one of these endonucleases will make a single cut that linearizes the circular plasmid  DNA , and allow it to recombine with foreign  DNA  that has been cut with the same endonuclease.

11. Cloning Vector pUC18 In the presence of IPTG, transformants expressing both fragments of the ßgalactosidase will form a functional enzyme and can be detected as blue colonies on agar plates containing X-Gal. Cloning into the multiple cloning site will lead to a nonfunctional N-terminal fragment of the ß- galactosidase and to the abolishment of α-complementation. White colonies will form on X-Gal/IPTG plates.

12. Cloning Vector pBR322

13. Cloning Vector pBR322 pBR322  is a  plasmid  and was one of the first widely used  E. coli   cloning   vectors . Created in 1977 in the laboratory of  Herbert Boyer  at the  University of California, San Francisco , it was named after the  postdoctoral researchers  who constructed it. The p stands for "plasmid," and BR for "Bolivar" and " Rodriguez." Cloning into the multiple cloning site will lead to a nonfunctional N-terminal fragment of the ß- galactosidase and to the abolishment of α-complementation pBR322 is 4361 base pairs in length  and has two antibiotic resistance genes – the  gene   bla  encoding the  ampicillin   resistance  ( Amp R )  protein , and the gene  tetA  encoding the  tetracycline  resistance ( Tet R ) protein. It contains the  origin of replication  of pMB1, and the  rop  gene, which encodes a restrictor of plasmid copy number. The plasmid has unique  restriction sites  for more than forty  restriction enzymes . Eleven of these forty sites lie within the Tet R  gene. There are two sites for restriction enzymes  HindIII  and  ClaI  within the  promoter  of the Tet R  gene. There are six key  restriction sites  inside the Amp R  gene .

14. Cloning Vector pBR322 The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the count increases through the Tet R  gene. The Amp R  gene is  penicillin   beta-lactamase . Promoters P1 and P3 are for the beta-lactamase gene. P3 is the natural promoter, and P1 is artificially created by the  ligation  of two different DNA fragments to create pBR322. P2 is in the same region as P1, but it is on the opposite strand and initiates  transcription  in the direction of the tetracycline resistance gene. A large number of other plasmids based on pBR322 have since been constructed specifically designed for a wide variety of purposes . Examples include the  pUC  series of plasmids.Most   expression vectors  for extrachromosomal protein expression and  shuttle vectors  contain the pBR322 origin of replication, and fragments of pBR322 are very popular in the construction of intraspecies shuttle or binary vectors and vectors for targeted integration and excision of DNA from chromosome

15. Cloning Vector Lambda phase

16. Cloning Vector Lambda phase Lambda phage was first isolated by Esther Lederberg in 1950 from Escherichia coli. It has been an intensely studied organism, and has been a useful tool in molecular biology. Lambda phage can be used for cloning of recombinant DNA, the use of its site specific recombinase ( int ) for the shuffling of cloned DNAs by the 'Gateway' method, and in recombineering . Bacteriophage Lambda was studied by Allan Campbell in 1962 who studied the phage's integration process . Lambda phage is a virus particle consisting of a head made up of double-stranded linear DNA as its genetic material, and a tail. It infects the host cell by injecting its own DNA through the tail at which point the phage will enter the lytic or lysogenic pathway. Large segments of the 48 kilobase pair DNA of the lambda phage are not essential for productive infection and can be replaced by foreign DNA, thus making lambda phage an ideal vector He observed that lambda phage had a unique characteristic what some phages would infect and reproduce in some strains of E. coli while other strains seemed immune.

17. Cloning Vector Lambda phase Its linear genome was observed to form a circle via complementary base pairing when entering a host cell. The genome was able to act in such a peculiar manner due to the presence of single stranded areas called cohesive sites ( cos ). These  cos  sites are able to base pair, and the nature of these sites are the same as those produced by  restriction endonucleases . (Ex:  Eco R1 produces "sticky ends"). This circular phage DNA is able to recombine with the host cell DNA via attachment sites. Progeny of the phage with the addition of some bacterial gene take place by the  lysogenic pathway . What makes lambda phage extremely advantageous is the fact that it can destroy its host ( lytic pathway ) or it can become part of its host ( lysogenic pathway ). Studies on the control of these alternative cycles have been very important for our understanding of the regulation of gene transcription.

18. Restriction: Phages grown in one bacterial cell fail to grow in other bacterial strains. The degradation of phage lambda DNA was observed in certain bacterial strains. This was later found to be a result of restriction endonucleases which would cut the phage DNA after detecting it as being foreign. Thus, restriction endonucleases was termed because of its ability to restrict foreign DNA. Modification: Phages are modified such that they can be grown normally in other bacterial strains. An example of modification is the methylation of lambda phage DNA so that it would not be cut by restriction endonuclease .

19. Tumor inducing plasmids (Ti plasmid) are double stranded circular DNA present in Agrobacterium tumefaciens . Agrobacterium is a Gram negative soil bacterium which infects dicotyledonous plants. This plasmid is denatured at higher temperatures and loses tumorgenic properties. Agrobacterium tumefaciens infects damaged plant tissues , induces the formation of a plant tumor called crown gall. The entry of bacterium in to plant tissues is facilitated by the release of certain phenolic compounds ( acetosyringone ) by the damaged site Ti Plasmid

20. Virulence Region Genes in the virulence region are grouped into the operons virABCDEG , which code for the enzymes responsible for mediating transduction of T-DNA to plant cells. virA codes for a receptor which reacts to the presence of phenolic compounds such as acetosyringone , which leak out of damaged plant tissues. virB encodes proteins which produce a pore/ pilus -like structure virC binds the overdrive sequence. virD1 and virD2 produce endonucleases which target the direct repeat borders of the T-DNA segment, vir E Binds to T-strand protecting it from nuclease attack , and intercalates with lipids to form channels in the plant membranes through which the T-complex passes , beginning with the right border. virG (TRANSCRIPTIONAL FACTOR) activates vir-gene expression after binding to a consensus sequence, once it has been phosphorylated by virA .

21. Application of Ti plasmid Plant transformation technique using Agrobacterium: Agrobacterium mediated technique is the most widely used for the transformation of plants and generation of transgenic plants. During transformation, several components of the Ti plasmid enable effective transfer of the genes of interest into the plant cells. These include: T-DNA border sequences, which demarcate the DNA segment ( TDNA) to be transferred into the plant genome. vir genes (virulence genes), which are required for transferring the T-DNA region to the plant but are not themselves transferred, and modified T-DNA region where the genes that cause crown gall formation are removed and replaced with the genes of interest.

22. BAC (Bacterial artificial chromosome )

23. RepE : for plasmid replication and regulation of copy number. ParA and parB : for partitioning F plasmid DNA to daughter cells during division and ensures stable maintenance of the BAC. Selectable marker: for antibiotic resistence ; some BACs also have lacZ at the cloning site for blue/white selection. T7 & Sp6: phage promoters for transcription of inserted genes. OriS : the origin of replication

24. APPLICATION OF BAC BACs are being greatly used in modeling genetic diseases in order to study their effects in the experimentation on transgenic mice. BAC have been used to study neurological diseases such as Alzheimer’s disease or in the case of down syndrome. The genome of several large DNA Viruses and RNA viruses have been cloned as BACs. These constructs are referred to as “ infectious clones ”.

25. YEAST ARTIFICIAL CHROMOSOME (YAC) First described in 1983 by Murray and Szostak . Yeast artificial chromosomes is a human engineered DNA molecule used to clone DNA sequences in yeast cells. YACs are shuttle vectors capable of replicating and being selected in common bacterial hosts such as E.coli as well as in the yeast S . cerevisiae . They are capable of carrying approximately upto 1000 kbp of inserted DNA sequence. Many different YAC plasmids exist, such as pYAC3 & pYAC4 .

26. COMPONENTS OF YAC The vector contains several elements of typical yeast chromosomes including: CEN: A yeast centromere, that ensures chromosome partitioning between two daughter cells and a selective marker genes. ARSI: Yeast autonomously replicating sequence TEL: Yeast telomere Yeast selectable marker such as URA3 & TRP1 and Bacterial selectable marker CONSTRUCTION OF YAC A YAC is built using an initial circular DNA plasmid, which is typically cut into a linear DNA molecule using restriction enzymes. DNA ligase is then used to ligate a DNA sequence or gene of interest into the linearized DNA, forming a single large, circular piece of DNA .

27. APPLICATION OF Y AC Appropriately modified YACs can be utilized in many different organisms, for cloning or genome analysis. Chromosomal translocation (chromosome abnormality that occurs due to rearrangement of parts among non homologous chromosomes) can be studied by means of disposable YACs that do not contain genetic informationnecessary for cell function. To identify essential mammalian chromosomal sequences necessary for the future construction of specialized mammalian artificial chromosomes(MACs).

28. M.MADHUSUDHANA REDDY Msc