Genetic Engineering. In biopharmaceutics

It's is a concept in bio pharmaceutics and 3rd year 3.2 semister topic this topic is from 1st unit in biopharmaceutics it is the last topic of 1st unit in biopharmaceutics

1. GENETIC ENGINEERING

2. Genetic engineering involves the manipulation of genetic material(DNA) This technique is used to achieve the desired goal in a pre-determined way Synonyms of Genetic engineering: - Gene manipulation - Recombinant DNA (r DNA) technology - Gene cloning (molecular cloning) - Genetic modifications - New genetics

3. HISTORY: In 1973, Boyer and Cohen performed experiments that involves recombination of two plasmids i.e., pSC 101 and pSC 102. The produced recombinant was introduced into E.coli and was found to possess both the characteristics of the parent molecules pSC 102 kanamycin resistant pSC 101 Tetracycline resistant Restriction by endonucleases and joining by ligases Recombinant plasmid shows both tetracycline and kanamycin resistance

4. The second set of experiments by Boyer and Cohen includes the production of frog protein in E.coli by using the frog DNA fragments and plasmid DNA fragments where the pairing occurred between complimentary DNA fragments frog protein from pSC 101 DNA Xenophs laevis Cut with ECoR1 to produce complimentary ends Addition of DNA ligase Recombined plasmid Introduced into E.coli and grown in nutrient rich medium Production of frog protein in E.coli

5. Hence by the above contributions made by these scientists, Biotechnologists divide the subject into two chronological categories namely BBC – Biotechnology Before Boyer and Cohen ABC - Biotechnology After Boyer and Cohen

6. STEPS IN GENETIC ENGINEERING: Generation of DNA fragments and selection of desired piece of DNA Insertion of selected DNA into a cloning vector to create a recombinant DNA or Chimeric DNA Introduction of recombinant vectors into host cells Multiplication and selection of clones containing recombinant molecules Expression of the gene to produce the desired product.

7. STEPS IN GENETIC ENGINEERING

8. GENERATION OF DNA FRAGMENTS: For molecular cloning, both the source DNA that contains the target sequence and the cloning vector must be consistently cut into discrete and reproducible fragments. This is achieved by the use of restriction endonucleases which cut the DNA at various sites. These are generally obtained from bacterial sources that can cut/ split DNA at specific sites. These enzymes are first discovered in E.coli that restricted the replication of bacteriophages. Eg. Eco RI NOMENCLATURE OF RESTRICTION ENDONUCLEASES: Eco RI Genus name ( Escherichia) Species name (coli) Strain (Ry 13) Roman numerical of order of discovery (I)

9. TYPES OF ENDONUCLEASES: TYPE I: A single enzyme with 3 sub units for recognition, cleavage and methylation. It can cleave upto 1000 bp from recognition site. Eg Ecok12 TYPE II: Two different enzymes either to cleave or modify the recognition sequence. Cleavage site is same or close to recognition site. Eg : EcoRI TYPE III: A single enzyme with 2 sub units for recognition and cleavage. Cleavage site is 24-26bp from recognition site. Eg : EcoPI TYPE IIs: Two different enzymes, cleavage site is upto 20bp from recognition site

10. RECOGNITION SEQUENCE: It is the site where DNA is cut by a restriction endonuclease Restriction endonucleases specifically recognize the particular sequences in DNA having 4-8 nucleotides and cleave. Each recognition sequence has a two fold symmetry i.e., the same nucleotide sequence occurs on both the strands of DNA that run in opposite direction. Such sequences are called as palindromes. CLEAVAGE PATTERNS: Restriction endonucleases cut DNA at definite sites within recognition sequence. The cleavage of DNA results in the formation of sticky ends (cohesive ends) or blunt ends

11. DNA LIGASES: The cut DNA fragments are joined together covalently by ligases These enzymes were first isolated from viruses, also found in E.coli and eukaryotic cells Ligases play an important role in cellular DNA repair process. Ligases seals the DNA fragments by forming a phosphodiester bond between the phosphate group of 5’ carbon of one deoxy ribose with the hydroxyl group of 3’ carbon of another deoxyribose. For the formation of phosphor diester bond a cofactor is required such as ATP for phage T4 DNA ligase and NAD + in case of E.coli DNA ligase The action of the DNA ligases is the final step for the formation of a recombinant DNA molecule.

12. HOMOPOLYMER TAILING: The complimentary DNA strands can be joined by a process called annealing. The technique involves the addition of oligo ( dA ) to the 3’ ends of some DNA molecules and the addition of oligo (dT) to the 3’ end of other molecules. The homopolymer extensions can be synthesized by using terminal deoxy- nucleotidyltransferase .

13. LINKERS AND ADAPTERS: These are chemically synthesized, short, double stranded DNA molecules. Linkers possess restriction enzyme cleavage sites that can be ligated to blunt ends of any DNA molecule and cut with specific restriction enzymes to produce sticky ends. Adapters contain preformed sticky or cohesive ends used to ligate DNA with blunt ends The DNA fragments held to linkers or adapters are ligated to vector DNA molecules

14. ALKALINE PHOSPHATASE: This is an enzyme responsible for removal of phosphate groups. This is used to prevent the unwanted ligation of DNA molecules When the linear vector plasmid DNA is treated with alkaline phosphatase, the 5’ terminal phosphate is removed This prevents both recircularization and plasmid DNA dimer formation

15. DNA MODIFYING ENZYMES: These enzymes participate in cutting and joining of DNA manipulation techniques. They can be classified into Nucleases Polymerases Enzymes modifying ends of DNA molecules

16. NUCLEASES: These enzymes cause the breakdown of phosphor diester bonds of DNA. They can be further categorized into endonucleases that act on internal phosphodiester bonds and exonucleases that degrade DNA at terminal end Eg : Endonucleases – nuclease S1 specifically acts on single stranded DNA and RNA molecules Deoxyribonuclease I cuts either single or double stranded DNA molecules at random sites Exonuleases – exonuclease III cuts DNA and generates molecules with protruding 5’ ends. Nuclease Bal 31is a fast acting 3’ exonuclease that couples with slow acting endonucleases

17. POLYMERASES: These are the enzymes that catalyse the synthesis of nucleic acid molecules. The enzyme is named after the nucleic acid template upon which it acts. Types of polymerases DNA dependent DNA polymerase – copies DNA from DNA RNA-dependent DNA polymerase --- also called as reverse transcriptase that synthesizes DNA from RNA DNA - dependent RNA polymerase --- produces RNA from DNA

18. ENZYMES MODIFYING THE ENDS OF DNA: These enzymes act on the terminal ends of DNA resulting in modification the molecule They include Alkaline phosphatase – removes the terminal phosphate group Polynucleotide kinase – adds phosphate groups to the DNA molecule Terminal transferase – also called as terminal deoxynucleotidyl transferase functions by repeated addition of nucleotides to 3’ terminal ends. It is widely used in homopolymer tailing.

19. 2. Selection of a vector: Vectors are the DNA molecules that can carry a foreign DNA to be cloned. These molecules possess self replicating capacity in an appropriate host cell Examples of vectors include plasmids, bacteriophages, cosmids and phasmids. CHARACTERISTICS OF AN IDEAL VECTOR: Small in size Should have a single restriction site An origin of replication 1-2 genetic markers for identification

20. PLASMIDS: These are extrachromosomal, double stranded, circular, self-replicating DNA molecules. These are present in almost all types of bacteria. They can be present in each cell ranging from 1-4 per cell (low copy number) to 10-100 per cell (high copy number). They vary in sized ranged from 1 to 500 kb Plasmid DNA contribute to about 0.5 to 5% of the total DNA of bacteria. Exceptions: A few bacteria such as belonging to genera Streptomyces sp , Borella burgdorferi contain linear plasmids.

21. TYPES OF PLASMIDS: There are various methods of classification of plasmids Method I: Basing on the presence of transfer genes Conjugative plasmids --- they contain a set of transfer genes facilitating bacterial conjugation Non-conjugative plasmids --- they don’t possess transfer genes Method II: Basing on the copy number Stringent plasmids --- present in limited number in cells (1-2) Relaxed plasmids --- occur in large number in each cell. Method III: Basing on the type of genes F-plasmids – possess genes for their own transfer from one cell to another R-plasmids – these carry genes resistance to antibiotics.

22. In general the conjugative plasmids are large, show stringent control of DNA replication and are present in low numbers. Non-conjugative plasmids are small, show relaxed control of DNA replication and are present in high numbers. NOMENCLATURE: They are designated by lower case letter ‘p’ followed by the first letters of the researcher’s name and numerical name given by them Eg : pBR322 stands for plasmid discovered by Bolivar and Rodriguez who designated it as 322 pUC101 is plasmid designed at University of California.

23. pBR322: Belongs to E.coli

24. BACTERIOPHAGES: These are the viruses that replicate in bacteria In certain phages during replication of DNA, part of their DNA gets incorporated into the bacterial chromosome and remains there permanently. Phage vectors can accept short fragments of foreign DNA into their genomes This makes the phages advantageous in carrying larger DNA fragments than plasmids. This makes the phage vectors suitable for working with human cells also.

25. BACTERIOPHAGES: Bacteriophage  or phage , a virus of E.coli is a widely used vector. Phage  consists of a head and tail where the DNA is located in the head which is a linear molecule of 50kb Upon its attachment to E.coli, phage injects its linear DNA into the cell that cyclizes and gets ligated through cos ends to form a circular DNA. The phage DNA may undergo two cycles namely Lytic cycle and Lysogenic cycle. The lytic cycle results in the release of phage particles after their synthesis and lysis of the cell at the end. Around 100 phage particles are produced within 20 min after the entry of phage into the bacterial cell

26. In the lysogenic type, the phage DNA becomes integrated into the E.coli chromosome and replicates along with the host genome. No phage particles are synthesized in this pathway. Importance of phage : Only about 50% of the phage DNA is required for its multiplication and other functions. As such the remaining of the phage DNA can be replaced by a donor DNA for cloning experiments. The packed phage containing foreign DNA can be injected into a host cell directly very effectively.

27. PHAGE M 13 VECTORS: These are single stranded DNA phage of E.coli. Inside the host cell, the vector synthesizes the complimentary strand to form a double stranded DNA(RF DNA). For use as a vector, RF DNA is isolated and a foreign DNA can be inserted on it which is then returned to the host cell as a plasmid. Single stranded DNAs are recovered from the phage particles

28. COSMIDS: These are the vectors that possess both the characteristics of both plasmids and bacteriophage . Cosmids can be constructed by adding a fragment of Phage  DNA including a cos site to plasmids. A foreign DNA about 40kb can be inserted into cosmid DNA. The recombinant DNA so formed can be packed as phages and injected into E.coli. Once they enter into the host cells, cosmids behave as plasmids and replicate. The advantage with cosmids is carrying capacity of foreign DNA which is more than plasmids.

29. PHASID VECTORS: These are also combination of plasmid and phage and can function as either one. Phasids possess functional functional origins of replication of both plasmid and phage  and can be propagated appropriately in E.coli

30. ARTIFICIAL CHROMOSOME VECTORS: NAME DESCRIPTION USES HUMAN ARTIFICIAL CHROMOSOME SYNTHETICALLY PRODUCED VECTOR DNA, POSSESSING CHARACTERISTICS OF HUMAN CHROMOSOME IT CAN CARRY TOO LONG HUMAN GENES ALSO USED IN GENE THERAPY YEAST ARTIFICIAL CHROMOSOME SYNTHETIC DNA THAT IS CONSIDERED AS LARGE CAPACITY VECTORS. THEY ALSO POSSESS CENTROMERIC AND TELOMERIC REGIONS USED TO CLONE LARGE PIECES OF DNA BACTERIAL ARTIFICIAL CHROMOSOMES (BACs) THEY ARE CONSTRUCTED F-PLASMID VECTORS. THEY HAVE GREATER STABILITY THAT YAC’s SEQUENCING OF HUMAN GENOME SHUTTLE VECTORS THESE ARE PLASMIDS THAT REPLICATE IN TWO DIFFERENT HOSTS. IN GENE CLONING

31. SELECTION OF HOST CELLS: The host cells are the living systems or cells in which the carrier of recombinant DNA molecule or the vector can be propagated. Different types of host cells suit different purposes namely Prokaryotic cells --- bacterial cells Eukaryotic cells – fungi, animal and plant cells Host cells help in effectively incorporate the vector DNA must be cultivated under appropriate conditions to collect the products. Among the various host cells, microorganisms are generally preferred as they multiply at a faster rate when compared to higher organisms

32. PROKARYOTIC HOST: The bacterium E.coli was the first organism used in DNA technology and continues to be the host of choice because of less doubling rate i.e., 20 min At the same time it suffers from limitations that include causation of diarrhoea by some strains, formation of endotoxins and low export ability of proteins. Also, post translational modifications are not possible in prokaryotic cells. Another microorganism Bacillus subtilis, a rod shaped non-pathogenic bacteria is widely used in the production of enzymes, antibiotics, insecticides etc., 2. EUKARYOTIC HOST: These hosts are used to produce human proteins The most commonly used host are yeast, Saccharomyces cerevisiae. Mammalian cells such as mouse cells are also employed as hosts as they have the post translational machinery that is lacking in prokaryotic cells

33. 4. INTRODUCTION OF RECOMBINANT VECTORS INTO HOST CELLS: Also called as gene transfer The efficiency of this process is crucial for the success of the technique. The commonly employed methods are Transformation Conjugation Electroporation Lipofection Direct transfer of DNA

34. TRANSFORMATION: Transformation is the method of introducing foreign DNA into bacterial cells (e.g. E.coli). The uptake of plasmid DNA by E.coli is carried out in ice-cold CaCl 2  (0-5°C), and a subsequent heat shock (37-45°C for about 90 sec). By this technique, the transformation frequency, which refers to the fraction of cell population that can be transferred, is reasonably good e.g. approximately one cell for 1000 (10 -3 ) cells. Transformation efficiency: It refers to the number of trans-formants per microgram of added DNA. For E.coli, transformation by plasmid, the transformation efficiency is about 10 7  to 10 8  cells per microgram of intact plasmid DNA. The bacterial cells that can take up DNA are considered as competent. The competence can be enhanced by altering growth conditions.

35. The mechanism of the transformation process is not fully understood. It is believed that the CaCI 2  affects the cell wall, breaks at localized regions, and is also responsible for binding of DNA to cell surface. A brief heat shock (i.e. the sudden increase in temperature from 5°C to 40°C) stimulates DNA uptake. In general, large-sized DNAs are less efficient in transforming. Sometimes, calcium phosphate may result in precipitate and toxicity to the cells. It is replaced with diethyl amino ethyl dextran (DEAE -dextran) for DNA transfer.

36. 2. Conjugation: Conjugation is a natural microbial recombination process. During conjugation, two live bacteria (a donor and a recipient) come together, join by cytoplasmic bridges and transfer single-stranded DNA (from donor to recipient). Inside the recipient cell, the new DNA may integrate with the chromosome (rather rare) or may remain free (as is the case with plasmids). Conjugation can occur among the cells from different genera of bacteria ( e.g Salmonella and Shigella cells). This is in contrast to transformation which takes place among the cells of a bacterial genus. Thus by conjugation, transfer of genes from two different and unrelated bacteria is possible. The natural phenomenon of conjugation is exploited for gene transfer. This is achieved by transferring plasmid-insert DNA from one cell to another. In general, the plasmids lack conjugative functions and therefore, they are not as such capable of transferring DNA to the recipient cells. However, some plasmids with conjugative properties can be prepared and used.

37. 3. Electroporation: Electroporation is based on the principle that high voltage electric pulses can induce cell plasma membranes to fuse. Thus, electroporation is a technique involving electric field-mediated membrane permeabilization. Electric shocks can also induce cellular uptake of exogenous DNA (believed to be via the pores formed by electric pulses) from the suspending solution. Electroporation is a simple and rapid technique for introducing genes into the cells from various organisms (microorganisms, plants and animals). The cells are placed in a solution containing DNA and subjected to electrical shocks to cause holes in the membranes. The foreign DNA fragments enter through the holes into the cytoplasm and then to nucleus.

38. Electroporation is an effective way to transform E.coli cells containing plasmids with insert DNAs longer than 100 kb. The transformation efficiency is around 10 9  transformants per microgram of DNA for small plasmids (about 3kb) and about 10 6  for large plasmids (about 130 kb). 4. Liposome-Mediated Gene Transfer: Liposomes are circular lipid molecules, which have an aqueous interior that can carry nucleic acids. Several techniques have been developed to encapsulate DNA in liposomes.

39. On treatment of DNA fragment with liposomes, the DNA pieces get encapsulated inside liposomes. These liposomes can adher to cell membranes and fuse with them to transfer DNA fragments. Thus, the DNA enters the cell and then to the nucleus. The positively charged liposomes very efficiently complex with DNA, bind to cells and transfer DNA rapidly. Lipofection is a very efficient technique and is used for the transfer of genes to bacterial, animal and plant cells.

40. 5.  Transduction: Sometimes, the foreign DNA can be packed inside animal viruses. These viruses can naturally infect the cells and introduce the DNA into host cells. The transfer of DNA by this approach is referred to as transduction. 6. Direct Transfer of DNA: It is possible to directly transfer the DNA into the cell nucleus. Microinjection and particle bombardment are the two techniques commonly used for this purpose. Microinjection: DNA transfer by microinjection is generally used for the cultured cells. This technique is also useful to introduce DNA into large cells such as oocytes, eggs and the cells of early embryos. The term transfection is used for the transfer DNA into eukaryotic cells, by various physical or chemical means.

41. 4. MULTIPLICATION AND SELECTION OF CLONES CONTAINING RECOMBINANT MOLECULES After the introduction of recombinant DNA into the cells it is necessary to identify the cells containing recombinant constructs. Various s election schemes are available for identifying cells with vector–insert DNA constructs. Transformed cells are distinguished from non-transformed cells by testing for resistance to specific antibiotics or by observing specifically colored colonies. Cells with a specific cloned target gene are identified by DNA hybridization with a homologous or heterologous probe, by immunological determination of an encoded recombinant protein, by the presence of a specified enzyme activity, or by functional (genetic) complementation4.4

42. Eg : Strategy for selecting host cells that have been transformed with pBR322. The transformation mixture, which contains three cell types, viz., nontransformed cells, cells with the intact original plasmid, and cells with DNA cloned into the BamHI site of pBR322, is plated on complete medium with ampicillin. The mixture is diluted beforehand to ensure separate colonies are formed on the agar. The nontransformed cells (Amps) are killed. The cells with the intact plasmid and cloned DNA–plasmid constructs are Ampr and therefore form colonies. Samples of the surviving colonies on the ampicillin plate are transferred to a plate with complete medium and tetracycline, keeping the same position of each colony on the second plate, i.e., replica plating. Only cells with intact plasmids ( Tetr ) will form colonies in the presence of tetracycline. The colonies that did not grow on the tetracycline plate (dashed circles) but grew on the ampicillin plate carry pBR322 with DNA that was cloned into the BamHI site. The colonies with cloned DNA inserts are picked from the original plate, pooled, and grown. The red square represents an orientation marker that keeps the master and replica plates aligned.

43. Screening bacterial colonies for mutant strains by replica plating. (A) Replica-plating (colony transfer) device; (B) replica-plating technique. Cells from each separated colony on a master plate (1) adhere to the velveteen of the replica plating device after it is gently pressed against the agar surface (2). The adhering cells are transferred (3), in succession, to a petri plate with complete medium (4)and to one with selective medium (5). The pattern of the colonies is consistent among the replicated plates because the orientation markers (red squares) are aligned for each transfer. In this example, minimal medium is the selective medium used to identify colonies that require a nutritional supplement for growth, i.e., auxotrophic mutants. The missing colony (dashed circle) on the minimal medium (5) denotes an auxotrophic mutation. The equivalent location on the plate with complete medium (4) has the colony with the auxotrophic mutation that can be picked and grown (6). Further analysis of the isolated strain is necessary to determine the nature of the auxotrophic mutation.

44. 5. Expression of the gene to produce the desired product After the isolation of the recombinants from the mixture of transformed and non-transformed cells, the cells are grown in a medium that supports the expression of the inserted genome sequence.