Genetic engineering Transformation .pptx

Genetics engineering

1. Genetic Engineering

2. Scope and History of Genetic Engineering Scope Construction of cloning and expression vectors Cloning of genes Studying gene function Production of novel proteins and enzymes Construction of DNA library Applications of genetic engineering Genetic transformation of plants Genetic transformation of animals Gene therapy Production of novel organisms with traits of interest

3. History Before 1950, experiments suggested DNA was genetic material. Early 1950s, DNA identified and structure discovered 1950s – early 60s, genetic code cracked, transcription and translation described However, laboratory techniques to study these novel genes stunk

4. 1997 Dolly cloned from the cell of an adult ewe DNA microarray technology developed 1998 The first animal genome ( Caenorharbditis elegans ) sequenced 1999 1274 Biotechnology companies in the United States 2000 Human Genome Project completed 2004 NIH launched GeMCRIS , an interactive web-based data allowing public access to information about gene transfer trials 2013 NIH revised rDNA guidelines to facilitate GE research

5. Early 70s, new methodology of recombinant DNA technology or genetic engineering involved use of gene cloning

6. Basics of Recombinant DNA Technology Gene Cloning Fragment of DNA inserted into a vector Results is a chimera or recombinant DNA molecule .

7. Vector carries gene into a host cell (usually bacteria) Gene Cloning

8. Chimera and host cell multiplies, creating many copies of the gene. Gene Cloning

9. Cell divisions create colony (clones) of identical host cells. Each cell contains one or more copies of recombinant DNA molecule Gene Cloning

10. Once gene is cloned, information can be gathered about its structure and expression Gene Cloning

11. If multiple DNA fragments are available, how is only one isolated? Gene Cloning

12. Usually, only one vector will enter a bacteria Gene Cloning

13. Q: How is the desired colony identified? A: Different ways; we will explore later Gene Cloning

14. Polymerase Chain Reaction Like gene cloning, PCR results in the amplification of a region of DNA PCR is not done in living cells PCR is done in test tubes with isolated DNA

15. Polymerase Chain Reaction PCR Hood

16. Thermal Cycler - amplifies DNA Polymerase Chain Reaction

17. Mixture of DNA and reagents (DNA polymerase & primers) heated to 95 ºC Polymerase Chain Reaction Heat breaks H-bonds; DNA denatures

18. Mixture is cooled (50-60 ºC), allowing primers to reanneal to DNA; based on primer sequence Polymerase Chain Reaction

19. Temperature increased (72-75 ºC), allowing DNA polymerase to attach and begin replication Polymerase Chain Reaction

20. Results in double stranded DNA fragments with primer sequences at their ends Polymerase Chain Reaction Cycle repeated 25-30 times

21. Gene Cloning vs. PCR In order to amplify an area, the sequences of the primer annealing sites must be known. Limited by length; regions >5 kb are difficult to amplify Limitations of PCR Gene Cloning is the only way of isolating long genes or unstudied genes

22. Gene Cloning vs. PCR PCR only amplifies one area

23. In order to clone a gene, it must be transported into living cell Vehicles for Gene Cloning Gene Cloning A DNA molecule needs to display several features to be used as a vehicle for gene cloning Most important features: replicate within the host cell small in size (< 10 kb)

24. Plasmids Bacteriophages Plant and animal viruses Transposons Artificial Chromosomes (BAC and YAC) Cosmids Phasmids Other advanced vectors

25. Two mechanisms for transport: That satisfy the criteria Plasmid – circular molecule of DNA Bacteriophages – protein capsule injects DNA Found in bacterial cells

26. Independent circular DNA in bacteria Plasmids Gene Cloning > Plasmids

27. Gene Cloning > Plasmids

28. Carry one or more genes Responsible for useful characteristics displayed by the host bacterium E.g. antibiotic resistance Gene Cloning > Plasmids

29. This antibiotic resistance is often used as selectable marker Gene Cloning > Plasmids When grown in antibiotic medium; only bacteria with resistant plasmid will grow

30. Have at least one origin of replication and the smaller one can use host cell’s enzymes to replicate independently The larger ones carry genes that code for special enzymes that are specific for plasmid replication Gene Cloning > Plasmids

31. Gene Cloning > Plasmids A few types of plasmids replicate by inserting themselves into bacterial chromosome Integrative plasmids or episomes

32. <10 kb useful for cloning Natural plasmids can range from 1 kb to 250 kb Only a few are useful for cloning purposes However, larger plasmids can be adapted for cloning under some circumstances Plasmid Size Gene Cloning > Plasmids

33. Copy Number Number of an individual plasmids per cell Range from 1 - >50 Ideally, higher copy number = better Gene Cloning > Plasmids

34. Conjugation and compatibility Plasmids fall into two groups: Conjugative Non-conjugative Conjugative plasmids are characterized by the ability to promote sexual conjugation between bacterial cells Gene Cloning > Plasmids

35. Conjugation allows for transfer of plasmid copy A non-conjugative plasmid may be co-transferred along with a conjugative plasmid

36. Gene Cloning > Plasmids Several different kinds of plasmids may be found in a single cell To be able to coexist in the same cell, different plasmids must be compatible If two plasmids are incompatible then one or the other will be quite rapidly lost from the cell

37. Fertility Plasmids – promote conjugal transfer of plasmids – carry tra genes Resistance plasmids – contain antibacterial resistance genes Col plasmids – code for colicins , proteins that kill other bacteria Plasmid Classifications Gene Cloning > Plasmids Based on the main characteristic coded by the plasmid genes

38. Degradative plasmids – allow host to metabolize unusual molecules Virulence plasmids – confer pathogenicity on host bacteria Plasmid Classifications Gene Cloning > Plasmids

39. Plasmids in organisms other than bacteria The best characterized eukaryotic plasmid is the 2 m circle that occurs in many strains of yeast This plasmid has allowed the construction of vectors for cloning genes with this organism as the host It is suspected that many higher organisms simply do not harbor plasmids within their cells

40. Viruses that specifically infect bacteria DNA (or RNA) surrounded by protein coat Genes code for capsid and replication proteins Bacteriophages Gene Cloning > Bacteriophages λ M13

41. Lytic Cycle: rapid infection resulting in lysis of cell and release of multiple bacteriophages Bacteriophages (Infection) Gene Cloning > Bacteriophages > λ

42. Phage attaches to outside of bacterium; injects DNA into cell Gene Cloning > Bacteriophages > λ

43. Phage genome replicated; with enzymes coded for by phage genome Gene Cloning > Bacteriophages > λ

44. Capsid proteins synthesized; phage particles assembled and released Gene Cloning > Bacteriophages > λ

45. Lysogenic phages : For bacteriophage λ , DNA integrates in to bacterium genome When released, enters lytic cycle Gene Cloning > Bacteriophages > λ

46. The integrated form of the phage DNA is called prophage The bacterium is referred to as lysogen Gene Cloning > Bacteriophages > λ

47. λ has “sticky ends”

48. A limited number of lysogenic phages follow a rather different infection cycle When M13 or a related phage infects E.coli , new phage particles are continuously assembled and released from the cell The M13 DNA is not integrated into the bacterial genome and does not become quiescent With these phages, cell lysis never occurs The infected bacterium can continue to grow and divide but at a slower rate than uninfected cells Gene Cloning > Bacteriophages > M13

49. Genes can be obtained in single strand form This is very useful when sequencing gene Much smaller than the λ genome = 6407 nucleotides Has room for more genes than λ genome Constructed from multiple copies of just three proteins requiring only three genes Synthesis of the λ head-and-tail structure involves over 15 different proteins Follow simpler infection cycle than λ M13 Advantage

50. Although there are many different varieties of bacteriophage , only λ and M13 have found a major role as cloning vectors

51. Viruses as cloning vectors Most living organisms are infected by viruses Therefore, viruses might be used as cloning vehicles for higher organisms

52. Attempts to use plant viruses as cloning vectors The potential of plant viruses as cloning vectors has been explored for several years but without great success One problem is that the vast majority of plant viruses have RNA genome Manipulations with RNA are difficult

53. Only two classes of DNA virus are known to infect higher plants The Caulimo viruses The Gemini viruses neither is ideally suited for gene cloning

54. Caulimovirus vectors In 1984, one of the first successful plant genetic engineering used a Caulimovirus vector to clone a new gene into turnip plants However, two general difficulties with these viruses have limited their usefulness

55. The total size of the genome Constrained by the need to package it into its protein coat This problem might be overcome by adopting helper virus strategy In this strategy, the cloning vector is a CaMV genome that lacks several of the essential genes

56. It can carry a large inserted gene but cannot itself direct infection Plants are inoculated with the vector DNA along with a normal CaMV The normal viral genome provides the genes needed for the cloning vector to be packaged into virus proteins and spread through the plant This approach has considerable potential, but does not solve the second problem

57. 2. Extremely narrow host range This restricts cloning experiments to just few plants, mainly brassicas such as: - turnips - cabbages and - cauliflowers

58. They have been important in genetic engineering as the source of highly active promoters that work in all plants Are used to obtain expression of genes introduced by Ti plasmid cloning or direct gene transfer

59. Geminivirus vectors Their natural hosts include maize and wheat Potential vectors for those and other monocots However, during the infection cycle the genomes undergo rearrangements and deletions Scrambles up any additional DNA They are starting to find specialist applications in plant cloning in the future

60. Cloning Vectors for Animals Cloning vectors based on insect viruses Baculovirus – played an important role in gene cloning with insects - use of this vector is in the production of recombinant protein

61. Cloning vectors for mammals Viruses were thought to be the key to cloning in mammals but only partially realized The first cloning experiment involving mammalian cells was carried out in 1979 with a vector based on Simian virus 40 (SV40) This virus is capable of infecting several mammalian species, following a lytic cycle in some and lysogenic cycle in others It also suffers from packaging constraints

62. Adeno viruses - enable larger fragments of DNA to be cloned than SV40 - but they are more difficult to handle because the genomes are bigger Papilloma viruses - enable to obtain stable transformed cell line Many mammalian viruses kill their host cells soon after infection, so special tricks are needed if these are to be used for anything other than short-term transformation experiments

63. Bovine papilloma virus (BPV) Causes warts on cattle Attractive because it has unusual infection cycle in mouse cells Takes the form of a multicopy plasmid with about 100 molecules per cell It does not cause the death of the mouse cell Passed to daughter cells on cell division Shuttle vectors consisting of BPV and pBR322 sequences Capable of replication in both mouse and bacterial cells Have been used for the production of recombinant proteins in mouse cell lines

64. At present, retroviruses are the most commonly used vectors for cloning genes in mammalian cells The most important application is in gene therapy

65. Cosmid Vectors Plasmids constructed with fragment of λ DNA including the cos site Packaging the cosmid recombinants into phage coats imposes a desirable selection upon their size With a cosmid vector of 5 kb, we demand the insertion of 32-47 kb of foreign DNA-much more than a phage- λ vector can accommodate

66. After packaging in vitro, the particle is used to infect a suitable host The recombinant cosmid DNA is injected and circularizes like phage DNA but replicates as a normal plasmid without the expression of any phage functions Transformed cells are selected on the basis of a vector-drug resistance marker They provide an efficient means of cloning large pieces of foreign DNA Attractive vectors for constructing libraries of eukaryotic genome fragments

67. Partial digestion provides suitably large fragments However, there is a potential problem associated with the use of partial digests in this way This is due to the possibility of two or more genome fragments joining together in the ligation reaction This creates a clone containing fragments that were not initially adjacent in the genome This would give an incorrect picture of their chromosomal organization The problem can be overcome by size fractionation of the partial digest

68. Even with sized foreign DNA, in practice cosmid clones that contain non-contiguous DNA fragments ligated to form a single insert may be produced The problem can be solved by dephosphorylating the foreign DNA fragments so as to prevent their ligation together This method is very sensitive to the exact ratio of target-to-vector DNAs because vector-to-vector ligation can occur Furthermore, recombinants with a duplicated vector are unstable and break down in the host by recombination Resulting in the propagation of a non-recombinant cosmid vector

69. Such difficulties have been overcome in a cosmid cloning procedure devised by Ish-Horowicz and Burke (1981) By appropriate treatment of the cosmid vector, left-hand and right-hand vector ends are purified which are incapable of self-ligation but which accept dephosphoreylated foreign DNA Thus the method eliminates the need to size the foreign DNA fragments and prevents formation of clones containing short foreign DNA or multiple vector sequences

70. An alternative solution to these problems has been devised by Bates and Swift (1983) who has constructed cosmid c2XB This cosmid carries a Bam HI insertion site and two cos sites separated by a blunt-end restriction site The creation of these blunt ends, which ligate only very inefficiently under the conditions used, effectively prevents vector self-ligation in the ligation reaction

71. Modern cosmids of the pWE and sCos series contain features such as Multiple cloning sites for simple cloning Phage promoters flanking the cloning site and unique Not I , Sac II or Sfi I sites (rare cutters) flanking the cloning site to permit removal of the insert from the vector as single fragments

72. BACs and PACs as alternatives to cosmids Phage P1 is a temperate bacteriophage which has been extensively used for genetic analysis of E. coli Its capacity is about twice that of cosmid clones but less than that of YAC clones The P1 vector contains a packaging site ( pac ) which is necessary for the in vitro packaging of recombinant molecules into phage particles

73. This P1 system has been used to construct genomic libraries of mouse, human, fission yeast and Drosophila DNA The vectors contain two lox P sites Recognized by the phage recombinase , the product of the phage cre gene Lead to the circularization of the packaged DNA after being injected into the E. coli host expressing the recombinase

74. BAC system has been developed for mapping and analysis of complex genomes Is based on the single-copy sex factor F of E. coli It includes the λ cos N and P1 lox P sites, two cloning sites ( Hind III and Bam HI ) and several G+C restriction enzyme sites for potential excision of the inserts

75. The cloning site is also flanked by T7 and SP6 promoters for generating RNA probes This BAC can be transformed into E. coli very efficiently BACs can maintain human and plant genomic fragments of over 300 kb for over 100 generations with a high degree of stability Have been used to construct genome libraries with an average insert size of 125 kb

76. Transposons In Drosophila P elements, which are one of several types of transposon 2.9 kb Contains three genes flanked by short inverted repeat sequences at either end of the element The genes code for transposase The inverted repeats form the recognition sequences that enable the enzyme to identify the two ends of the inserted transposon The vector is a plasmid that carries two p elements, one of which contains the insertion site for the DNA that will be cloned

77. Insertion of the new DNA into this p element results in disruption of its transposase gene The second p element carried by the plasmid is therefore one that has an intact version of the transposase gene Ideally this second element should not itself be transferred to Drosophila chromosomes, so it has ‘wings clipped’ Its inverted repeats are removed so that the transposase does not recognize it as a real p element Once the gene to be cloned has been inserted into the vector, the plasmid DNA is microinjected into fruit fly embryos

78. The transposase from the wings-clipped p element directs transfer of the engineered p element into one of the fruit fly chromosomes If this happens within a germline nucleus, then the adult fly that develops from the embryo will carry copies of the cloned gene in all its cells The same principle can be applied to other organisms

79. Artificial Chromosomes Yeast Artificial Chromosome (YAC) A totally new approach to gene cloning The development of YAC has been based on the key components of a chromosome structure Centromere Required for the chromosome to be distributed correctly to daughter cells during cell division 2. Two telomeres - The structures at the ends of a chromosome, which are needed in order for the ends to be replicated correctly Prevent the chromosome from being nibbled away by exonucleases

80. 3. The origins of replication - Are the positions along the chromosome at which DNA replication initiates - Similar to the ori of a plasmid

81. The structure and use of YAC vector Several YAC vectors have been developed Each one is constructed along the same lines pYAC3 is a typical example - It is essentially a pBR322 plasmid into which a number of yeast genes have been inserted - Two of these genes have been encountered as the selectable markers for yeast integrative plasmid (YIP5) and yeast replicative plasmid (YRp7)

82. - For YIp5 = URA3-codes for orotidine-5’-phosphate decarboxylase - An enzyme that catalyzes one of the steps in the biosynthesis pathway for pyrimidine nucleotides For YRp7 = TRP1-involved in tryptophan biosynthesis The DNA fragment that carries TRP1 also contains an ori , but in pYAC3 this fragment is extended even further to include the sequence called CEN4 The TRP1-ori-CEN4 fragment therefore contains two of the three components of the artificial chromosome

83. The third component, the telomeres, is provided by the two sequences called TEL - These TEL are not themselves complete telomere sequences - But once inside the yeast nucleus, they act as seeding sequences onto which telomeres will be built - SUP4 which is the selectable marker into which new DNA is inserted during the cloning experiment

84. The yeast strain that is used is a double auxotrophic mutant trp1 - ura3 - , which is converted to trp1 + ura3 + by two markers on the artificial chromosome Transformants are therefore selected by plating onto minimal medium, on which only cells containing a correctly constructed artificial chromosome are able to grow Any cell transformed with incorrect artificial chromosome is not able to grow on minimal medium as one of the markers is absent

85. The presence of the insert DNA in the vector can be checked by testing for insertional inactivation of SUP4, which is carried out by a simple color test Red colonies = recombinants White colonies = not recombinants

86. The cloning strategy with pYAC3 The vector is first restricted with a combination of Bam HI and Sna BI Cutting the molecule into three fragments The Bam HI fragment is removed, leaving two arms, each bounded by one TEL sequence and one Sna BI site The DNA to be cloned, which must have blunt ends is ligated between the two arms, producing artificial chromosome Protoplast transformation is then used to introduce the artificial chromosome into S. cerevisiae

87. Choice of Vector The size of insert is not the only feature of importance The absence of chimeras and deletions is even more important In practice, some 50% of YACs show structural instability of inserts or are chimeras Cosmid inserts sometimes contain the same aberrations The greatest problem with them arises when the DNA being cloned contain tandem arrays of repeated sequences

88. Potential advantages of the BAC and PAC systems over YACs Lower levels of chimerism Ease of library generation Ease of manipulation and isolation of insert DNA BAC clones seem to represent human DNA far more faithfully than their YAC or cosmid counterparts Excellent substrates for shotgun sequence analysis Accurate contiguous sequence data

89. Specialist-purpose vectors Vectors to make single-stranded DNA for sequencing Whenever a new gene is cloned or a novel genetic construct is made, it is usual practice to sequence all or part of the chimeric molecule Sanger method of sequencing requires single-stranded DNA as a starting material Originally, single-stranded DNA was obtained by cloning the sequence of interest in an M13 vector Today, the sequence is cloned into a pUC -based phagemid vector which contains the M13 ori region and the pUC (Col E1) ori Such vectors replicate inside the cell as double-stranded molecules

90. Single-stranded DNA for sequencing can be produced by infecting cultures with a helper phage such as M13K07 This helper phage has the ori of P15A and a kanamycin resistance gene inserted into the M13 ori region It carries a mutation in the gII gene M13K07 can replicate on its own In the presence of a phagemid bearing a wild-type ori , single-stranded phagemid is packaged preferentially and secreted into the culture medium DNA purified from the phagemids can be used directly for sequencing

91. Expression vectors Special vectors for expression of foreign genes Required to prepare RNA probes from the cloned gene or to purify large amounts of the gene product In these cases, transcription of the cloned gene is required It is usual to utilize a promoter specific to the vector Such vector-carried promoters have been optimized for binding of the E. coli RNA polymerase and many of them can be regulated easily by changes in the growth conditions of the host cell

92. RNA polymerase recognizes different types of promoters depending on which type of σ factor is attached The most common promoters are those recognized by the RNA polymerase with σ 70 A large number of σ 70 promoters from E. coli have been analysed A comparison of these promoters has led to a formulation of a consensus sequence If the transcription start point is assigned +1 then this consensus sequence consists of the -35 region (5’-TTGACA-) and the -10 region, Pribnow box (5’-TATAAT)

93. RNA pol must bind to both sequences to initiate transcription The strength of a promoter depends on how close its sequence is to the consensus The -35 and -10 regions are the sites of nearly all mutations affecting promoter strength Other bases flanking these regions can also affect promoter activity The distance between the -35 and -10 regions is also important The promoter is weaker when the spacing increases or decreases from 17 bp

94. Upstream (UP) elements located 5’ of the -35 hexamer in certain bacterial promoters are A+T-rich sequences That increase transcription by interacting with the  subunit of RNA pol Once RNA pol has initiated transcription at a promoter, it will polymerize ribonucleotides until it encounters a transcription-termination site in DNA