Lecture two Genetics engineering pptE.pptx

Genetics engineering in the medical field

1. Basic concepts of Genetic Engineering (GE) CHAPTER - TWO THE METHDOLOGY OF GENE MANIPULATION I) Host cell types II) Cloning and expression vectors in GE Plasmid Bacteriophages λ and M13 Other vectors (Hybridplasmid/phage, vectors in use in Eukaryotic cells, Arteficial chromosomes (YAC, BAC and others) III) rDNA delivery methods BY Dereje Beyene (Ph.D)

2. HOST CELL TYPES Here, host cell contextually refers to the recipent of the engineered/recombinant DNA/chimeric DNA (It encompass both prokaryot and eukaryotic cell types) The types of host cell selection will depend upon on the objective of the cloning procedure: If the aim is to isolate a gene and sequence for structural analysis . You may pick a simple host cell (e.g. E. coli ) and cloning vector OR for functional analysis you may pick a mutant E. coli or other relevant mutant and complement the mutation and the vector will be expression Vector If the aim is to express the genetic information in a higher eukaryote such as a plant, a more specific system will be required   N.B : These two aims are not mutually exclusive. E.g . Transgenic plant technology combines the two aims for successful introduction of the chimeric DNA into the plant system for improving the desired trait/metabolic modification.

3. II) Cloning and expression vectors in GE

4. I) Plasmid Biology: A Brief Overview Plasmids are autonomously replicating extra-chromosomal DNA elements ( replicons ) that are commonly present in bacteria, archaea , and yeasts. They often carry genetic information that is useful only under a typical conditions, and are sometimes lost from the cell when in more commonly encountered (non-selective) environments. Table 1 . Features of typical plasmids

5. Plasmid biology con’t STRUCTURE Plasmids can be circular or linear and range in size from smaller than one kilobase (Kb) to more than a megabase (Mb). REPLICATION Replication of plasmids is dependent primarily on enzymatic machinery of the host; however key controlling features are generally encoded by the plasmid . Using a variety of mechanisms - depending on the element – Plasmids regulate the frequency of replication initiation by one or more "Rep“ functions at a site called the origin of vegetative replication ( oriV ). oriV affects a characteristic copy number; the more frequent the initiation of a round of replication within a bacterial division cycle, the more copies per cell. PLASMID TRANSFER An important feature of many plasmids is the ability to transfer a copy of themselves from one bacterial cell to another upon cell-to-cell contact, a phenomenon known as conjugation ( Pls read how that happen? ). There are two types of plasmid based on self transfer ability: Conjugative plasmid – self transferable by using their Ter and mob region in their genome by the process of conjugation Non-conjugative plasmid – are not self transmissible but may be mobilized by conjugation-proficient plasmid if theitr’mob’ region is functional

6. Table 1. Features of some naturally occuring plasmids For purpose of GE the naturally occuring plasmids: The naturally occuring plasmids have been extensively modified to produce vectors that have the desired chracterstics. The vector could be cloning or expression vector. Plasmids naming: P is used to designate plasmid and usually follow the i nitial worker(s) who isolated or constructed the plasmid. The numbers may be used to describe the particular isolate. E.g. pUC : p plasmid and UC University of California

7. Plasmid biology con’t Plasmid Vector: should consiste of MCS, Selection marker, Ori New plasmid vectors can be constructed by rearranging various parts of the plasmid. This involves addition or deletion of DNA to change the charactestics of the vector. The hatched region shows the fragment that was removed from pBR322 to gnerate pATI53

8. Applications of plasmids Cloning and analysis of a variety of genes from a huge spectrum of biological sources. Commercial production of numerous biological agents (enzymes, vaccines, hormones, macromolecules useful in biotechnology, etc.) " Biofactories ". Constructing genomic and cDNA libraries those are useful for sequencing of entire chromosomes/ trancriptome of higher systems, including the complete human genome Sequencing of bacterial genomes P lasmids also played key roles in the genetic engineering of both plants and animals and have been important tools in gene therapy.

9. Cloning vectors : gene cloning, sequencing, Genomic/cDNA libraray construction Applications of plasmids con’t MCS increases the flexibility of vectors by providing a range of REs option for cloning

10. Applications of plasmids con’t Expression vectors : biological function validation, biofactory

11. The Biology of Bacteriophages λ P hages are viruses that can infect bacteria.  The major advantage of the phage vector is its higher transformation than the plasmid vector. Head + tail code B2 region Rce genes Regulation genes A) Linear Form OP – DNA synthesis RS – Host lysis Cos (Sticky) ends : single stranded DNA (12 nt). One end of the lilnear form is complementary to the other end thus the circular form can be made. B2 region : Not essential for survival. Rec-genes: Involved in recombination. Regulation genes: Genes that switch on/off early in process. B) Circular Form Basic Features of Bacteriophage λ

12. Fig X. The three types of capside structure common in bacteriophages Fig X. The lysogenic infection cycle, as followed by structure common in bacteriophage λ Bacteriophages λ con’t

13. Fig. X Map of the phage λ genome. Some of the genes are indicated. Functional regions are shown by horizontal lines and annotated. The non-essential region that may be manipulated in vector construction is shaded Replaceable region Bacteriophages λ con’t

14. Bacteriophages λ con’t The non-essential region can be deleted without impairing the function required for the lytic infection cycle The wild-type λ phage will have generally multiple of RE’s recognition sites could be used for cloning. (This can be a major problem, as it limits the choice for site for insertion of foreign DNA and also it may be in the essential region of the viral genome map!) The wild-type λ phage with a reduced number of RE’s recognition sites will be selected ( This will be done by sequencing and restriction mapping ) A technique of in vitro mutagenesis will be used to modify the unwanted RE’s recognition sites Finally, It is possible to construct phage that have the desired combination of RE’s recognition sites. (i.e. Bacteriphage λ cloning vector is constructed )

15. RE RE Bacteriophages λ vector Fig. X . Schematic drawing of the DNA cloning using phages as vectors.  The DNA to be cloned is first inserted into the DNA, replacing a nonessential region.  Then, by an in vitro assembly system ( described below ), the virion carrying the recombinant DNA can be formed. 

16. Invitro packaging of λ cloning vector Packaging requires a numbar of proteins encoded by the λ genome, but these can be prepared at a high concentration from cells infected with defective λ phage strains. Two differnet systems are in use: 1) Single strain system & 2) Double strain System (Based on the number of E. coli (host) strains used) Single strain system: the defective λ phage carries a mutation in the cos sites, so that the endonuclease recognition sites are not recognized by the endocuclease that normally cleave the λ catenanes during the phage replication. Function of cos sites: Circularization of the λ genome in the host ( E. coli ), which is a prerequisite for insertion into the bacterial genome It has also a role after the λ genome excised from the host genome. The large number of the new λ DNA are produced by the rolling circle mechanism of replication , in which a continuous DNA strand is ‘rolled off’ the template molecule. The result is a catenane consisting of a series of linear λ genomes joined together at the cos sites. The role of the cos sites is now to act as recognition sequences for the endonucleases that cleaves the catanane at cos sites, producing individual λ genomes. This endonuclease , creates the single-stranded sticky ends, and also acts in conjunction with other proteins to package each λ genome into a phage head structure.

17. Mutation at cos sites: The defective phage can not replicate, though it does the direct synthesis of all the proteins for packaging. The protein accumulate in the bacterium and can be purified from cultures of E. coli infected with the mutated λ and used for in vitro packaging of recombinant λ molecules. ( One strain packaging system ) A) Two strain packaging system B) One strain packaging system E. Coli SMR10 - λ DNA has defective cos sites

18. Insertional phage vector Replacement phage vector Bacteriophages λ vector con’t ( 37Kb – 52Kb)

19. Bacteriophages λ vector con’t Fig. X Cloning with insertion λ vector. Assuming the packaging is effective if the size is 37- 52 Kb long. The short one ( i.e with out the insert) is < 37 Kb, the combination will not packed even if it carries the cos sites Packaging into the head of the virus is possible, if Cos sites should exist at both ends The total size of the genome 37 – 52 Kb (the cloning λ vector is less than 37 ), the size of self- ligated construct (both in replacment and insertional vectors) is less than 37 Kb

20. M13 Phage M13 is a flamentous phage M13 is a ssDNA genome, it is much smaller than the λ genome (M13 is 6407 nt in length) The virus inject its ssDNA (genome) into E.coli via pillus , then the ssDNA used as a template resulting in normal dsDNA ( i.e Replicative form, RF) dsDNA of the phage is not inserted into the host genome unlike bacteriophage λ. The Replicative form (RF) multiply >100 copies When the host bacterium divides, each daughter cell receive the copies of the phage genome The new particle is assembled and released about 1000 new phages being produced Fig. X. M13 structure, with the ssDNA genome being enclosed in a protein coat. The gene3 product is important in both adsorption and extrusion of the phage .

21. Vectors based on bacteriophage M13 The genome of M13 is <10Kb in size, it is well within the range of desirable size for a potential vector. dsDNA (RF) of M13 genome behaves very much like a plasmid M13 does not have any non-essential genes, deleted to increase the size of insert during cloning , so that the only part avilable for manipulation is a 507 bp intergenic region Thus, marked reduction in cloning effeciency compared to λ ( about 20 Kb), where the M13 is limited about 1.5Kb This has been used to construct the M13mp series of vectors (e.g. Fig X)It is easely prepared from a culture of infected E. coli and can be re- introduced by transinfection Gene cloned into M13 based vector can be obtained in the form of ssDNA. Thus, ssDNA is one of a usefull tool for Sequencing and site directed mutagenesis

22. Bacteriophage M13 con’t Fig. X. Map of M13 18. The dsDNA RF is shown. The MCS is the same that is found in pUC18. The vector M13mp 19 is identical except for the orientation of MCS (Encode protein for capside formation) (Encode protein for phage morphogenesis) (Encode DNA replication protein)

23. Other vectors Why? As cloning methdology developed, plasmid- and phage based vectors placed some constraints. The major limitations are the cloning capacity and stability of the insert These limitations initiated for inovation of other vectors with features such as Insert stability Higher cloning capacity that surpass maximum limit of phage and plasmid Cloning capacity. Vector that could infect eukaryotic host cells. Other vectors invented are : Cosmids and phagemids Arteficial chromosomes (AC) (Yeast AC; YAC, Bacteria AC; BAC....

24. Types of vector Insert size (Kb) No = clones* P = 95% No = clones* P= 99% λ vector 18 532,500 820,000 P1 125 77,000 118,000 Cosmid, Fosmid 40 240,000 370,000 BAC, PAC 300 32,000 50,000 YAC 600 16,000 24,500 Mega-YAC 1400 6850 10,500 * Calculated from the equation: N= ln(1-p)/ln(1-a/b) N = the number of colonies required P= probability that any segment of the genome is present in the libraray a = average size of DNA fragments inserted into the vector and b = the size of genome Table X. Sizes of human genomic libraries prepared in different types of cloning vector

25. Hybrid plasmid/phages vectors C osmid vector It is a combination of the plasmid vector and the cos site bacteriophage of λ ( cos site is the only part needed for enzymatic packaging of the phage in to the head protein!) . Moreover, the in vitro packaging reaction works not only with λ genome, but also with any molecule that carries the cos sites separated by 37-52Kb of DNA!   It has the following advantages : A) High transformation efficiency. B) The cosmid vector can carry up to 40 kb whereas plasmid and phage vectors are limited to 25 kb.

26. Eco RI, Bam HI, Hin dIII, Eco RIV....... Restrict with Bam HI cos Bam HI amp r ORI Bam HI Linear Cosmid New DNA/ Insert Bam HI Bam HI (Ligation, DNA ligase) concatamer In vitro packaging Fig. X Cloning by using cosmid vectors.    A) Cosmid vectors cleaved with Bam HI and B) The new DNA fragments with Bam HI recognition sites ligated .   Remark: The subsequent assembly and transformation steps are the same as cloning with phages. (A Recombinanat Cosmisd DNA λ particles

27. Yeast Artificial chromosome (YAC) Artificial chromosomes are elegant simple vectors that mimic the natural construction of chromosomal DNA of eukaryotes. It is the first breakthhroug in cloning > 50Kb achieved by YAC The features of eukaryotic chromosomes added in artificial chromosomes are (Fig): In YAC, the DNA sequences underlie these chromosomal componenets are linked together with one or more selectable markers and at least one restriction enzyme recognition site into which the new DNA inserted. All componenets of YAC can be contained in 10 – 15 Kb, natural yeast chromosomes range in size from 230 Kb to over 1700 Kb, so YACs have the potential to clone a Megabasepair sized DNA fragment. The potential of YAC to clone standard 600 Kb and special 14,000Kb Small arm Large arm

28. Working with YAC Steps in a gene of interest into cloning vector pYAC3 The circular vector is digested with BamHI and SnaBI. BamHI removes the stuffer fragment held bln the two tolemers in the circular molecule SnaBI cuts within the SUP4 gene and inactivate the gene activity (insertional inactivation) Ligation of the new DNA molecule at the two arms: the ligation product carries two functional genes ( TRP and URA3 ) and one inactivated SUP4 genes of selection markers The host strain has inactivated copies of the selection markers (TRP and URA3) which means that it requires tryptophan and uracil as a nutrient. The transformant are plated onto a minimal medium lacking TRP and URA3, are able to survive on this medium and produce colonies. If the a vector comprises two right arms, or two left arms , then it will not give rise to colonies blc the transformed cells will still require one of the nutrients.

29. Identification of the Recombinant yest The cloning site is within the SUP 4 gene, It suppresses a mutation at the ade2 locus in the host, resulting in a color change from red to white in the presence of limiting concentrations of adenine. ade2 locus encodes : Phosphoribosylaminoimidazole carboxylase , catalyzes a step in the 'de novo' purine nucleotide biosynthetic pathway; red pigment accumulates in mutant cells deprived of adenine This provides a convenient phenotypic characterization: If interruption of the SUP 4 gene occurs, recombinants will grow red and Non- recombinants with an intact suppressor will grow white

30. Limitations of YAC and other alternative cloning vectors Standard YAC can clone an insert size about 600 Kb and specialized mega YAC upto 14,000 kb, this is the highes capacity of any of the cloning vectors. Hence, several of the early genome projects made extensive use of YAC. Limitation: Some YAC cloning vectors has a problem of insert instability, the cloned DNA becoming re-arrenged into new sequence combinations. Because of the limitation, there is a great interest in other type of vectors, once that can not clone such large piece of DNA but which suffer less from instability problems. These vectors include: Bacterial Arteficial chromosomes (BAC) Cosmid Fosmid Bacteriophage P1 vector P1-derived arteficial chromosomes (PAC)

31. Bacterial artificial chromosmes (BAC) It is based on the naturally occuring F-plasmid of E.coli F-plasmids are large unlike other plasmids, vectors based on F-plasmids has higher capacity for accepting inserted DNA BAC are designed so that recombinanats can be identified by Lac selection (Fig.), hence easy to use They can clone fragments of 300 Kb and longer and the insert are very stable about 100 generations tested (e.g. pBAC108L) N.B: BACs are extensively used in human genome projects

32. Construction of BAC Vector Fig. X. BAC vector construct. 97 bp long synthetic nt sequence for T7/SP6 promoters, Two cloning sites were inseted into pMBO131, a 400-bp sac I fragment of bacteriophage λ carrring the cos N site was inserted into the sac I site of the plasmid. A 42 bp oligo nts having a bacteriophage P1 loxP site was inserted at Cal I site bln cos N and cloning site. (Chloroamphenicol resitant gene) The F plasmid not only codes for genes that are essential to regulate its own replicatiion and also control its copy number. The regulator genes include oriS , repE , parA, and parB. OriS and repE genes mediate the unidirectional transfer of the F factor while parA and parB maintain the copy number at the level of one or two per E.coli genome A loxP site (locus of X- ing over) consists of two 13 bp inverted repeats separated by an 8 bp asymmetric spacer region:

33. Fosmid They contain the F-plasmid origin of replication and a λ cos site. They are smaller than cosmid but have a lower copy number in E. Coli, which means they are less prone to instability problem. Bacteriophage P1 vector They are very similar to λ vectors, being based on the deletion of a natural phage genome, the capacity of the cloning vector being determined by the size of the deletion and the space bln the phage particle. P1 genome is larger than the λ genome, and the phage particle is bigger, so a P1 vector can clone large fragment then a λ vector, up to 125 Kb using current technology P1 derived artificial chromosomes (PAC) Combine features of P1 vectors and BAC, and have a capacity of up to 300 Kb

34. III) DNA delivery method

35. UP Take of exogenous genetic material In microbiology , genetics , cell biology and molecular biology , competence is the ability of a cell to take up extracellular ("naked") DNA from its environment. Competence may be differentiated between natural competence , a genetically specified ability of bacteria which is thought to occur under natural conditions as well as in the laboratory, and induced or artificial competence , which arises when cells in laboratory cultures are treated to make them transiently permeable to DNA. Chemical methods: 1) CaCl 2 (0.1M) method It is one of the chemical method, E.coli cell mebrane becomes transiently permeable to up take an Exogenous DNA when the competant E.coli subjected for short heat shock (42 ℃ ) about 30 to 40 sec. This enables us to tranform E.coli with various constructs generated using the state art of Genetic engineering 2) CaPO 4 Method: An animal cell can be directly treated with the chemical whereas, Plant, fungi cells, their cell wall is removed by enzymatic treatment (Protoplast)

36. Physical methods A) Micro-injection The host cell is immobilized by applying a mild suction with a blunt pipette. The foreign gene is then injected with a micro-injection needle. It does not have species specificity! Electroporation Temporary holes are formed in the plasma membrane of the host cell by applying a high voltage (8 kV/cm, 5µs). These pores permit entry of foreign DNA.

37. B) Biolistic method Its a mechanical method of DNA delivery method. The DNA is coated onto microprojectiles, which accelerated by the microprojectile on firing the gun The microprojectile is retained in the chamber and the shooting is carried on to the target tissue It needs technical skills It does not have species specificity!

38. Liposomes as a gene delivery sysyem Liposomes were discovered in 1961 by Alec D. Bangham who was studying phospholipids and blood clotting, and since then they became very versatile tools in biology, biochemistry and medicine. Definition: Liposomes are nano size artificial vesicles of spherical shape that can be produced from natural phospholipids and cholesterol. The lipid bilayer (similar to the cell membrane) of the liposome can fuse with other bilayers (e.g. cell membrane), thus delivering the liposome contents. It is a important tool for gene therapy and drug delivery (p harmaceuticals ) Transfection of the DNA DNA is packaged in vitro into phage particles phages are allowed to infect bacterial cells term also used in DNA transfer to eukaryotic cells DNA is transiently expressed

39. Liposome Con’t Steps for the successful trasfer in vitro involves: The packaging of DNA The adhesion of packaged DNA to the cell surface Internalization of DNA Escape of DNA from endosomes if endocytosis is involved DNA expression in cell nuclei