Cloning and Recombinant DNA PDF

Cloning and Recombinant DNA Lecture 10 Molecular Biology Department of Biology Faculty of Mathematics and Natural Sciences Universitas Indonesia  Principle of cloning Contents Restriction enzyme...

Cloning and Recombinant DNA Lecture 10 Molecular Biology Department of Biology Faculty of Mathematics and Natural Sciences Universitas Indonesia  Principle of cloning Contents Restriction enzyme Host cell Vector Promoter Transforman selection At a glance Less-flatulent cows Venomous cabbage Web-spinning goats What is Genetic Engineering? The process by which pieces of DNA are transferred from one organism to another. Enabling technology that involves cutting, modifying, and joining DNA molecules using enzymes such as DNA Often referred as gene ligase and restriction enzymes. manipulation, gene cloning, recombinant DNA technology, genetic modification, new genetics. Basic Steps: gene cloning Restriction Enzyme DNA Ligase Basic Steps: gene cloning a. DNA source is isolated and fragmented into suitably sized pieces. b. Fragments are joined to a carrier molecule (vector) to produce recombinant DNA molecules (in pic is plasmid) → become replicons. c. Recombinant DNA molecules are then introduced into host cell (shown: bacterial cell) for propagation. 3 things that need to be done to isolate a gene from a collection of recombinant DNA sequences: ▪ Separating every individual recombinant molecules ▪ Amplifying recombinant sequences to provide enough material for further analysis ▪ Selecting specific fragment by sequence-dependent method Gene cloning Gene cloning is achieved by using a suitable carrier molecule (vector) to propagate the desired sequence in a living host cell.  Principle of cloning Contents Restriction enzyme Host cell Promoter Vector criteria and types Mobile DNA Transforman selection Restriction enzymes are molecular scissors. A restriction enzyme (or restriction endonuclease) is an enzyme that cuts DNA at specific recognition nucleotide sequences known as restriction sites. They can be found in bacterial cells, acting as protection mechanism against exogenous DNA by hydrolising it. The bacteria’s endogenous DNA is not hydrolised because they are methylated.  How do bacteria prevent restriction enzymes from damaging its own DNA? Methylation can also occur in eukaryotic (plant) DNA Methylation may prevent gene expression,and may play a role in tissue specific gene expression Transgene silencing often associated with methylation  Types of Restriction Enzymes Restriction enzymes are categorized into four general groups. These types are categorization based on: composition. enzyme co-factor Type Type requirement. I II the nature of their target sequence. position of their DNA Type Type cleavage site relative to III IV the target sequence.  Types of Restriction Enzymes Type Characteristics Cleave at sites remote from recognition site; require both ATP and I S-adenosyl-L-methionine to function; multifunctional protein with both restriction and methylase/methyltransferase activities. Cleave within or at short specific distances from recognition site; II most require magnesium; single function (restriction) enzymes independent of methylase. Cleave at sites a short distance from recognition site; require ATP (but do not hydrolyse it); S-adenosyl-L-methionine stimulates III reaction but is not required; exist as part of a complex with a modification methylase. Enzymes target modified DNA, e.g. methylated, IV hydroxymethylated and glucosyl-hydroxymethylated DNA.  Nomenclature Restriction enzymes named after the bacterium from which they are purified 1. First letter from genus 2. Next two letters represent species 3. Additional letter or number represent the strain or serotypes. HindII Haemophilus influenzae EcoRI E. coli PstI Providencia stuartii NotI Norcardia otitidiscaviarum AluI Arthrobacter luteus  Restriction site ▪ Also called cleavage site or restriction endonuclease site. ▪ The specific nucleotide site recognised by restriction endonuclease. ▪ While recognition sequences vary widely, with lengths between 4 and 8 nucleotides, many of them are palindromic.  Palindromic Sequence ▪ The mirror-like palindrome in which the same forward and backwards are on a single strand of DNA strand, as in GTAATG. ▪ The inverted repeat palindrome is also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands (GTATAC being complementary to CATATG). ▪ size of recognition sequence determines frequency of cleavage ▪ 1/44 = every 256 bp on average ▪ 1/46 = every 4096 bp on average.  Function & Mechanism ▪ Function: Restriction enzymes protect cells from foreign DNA(they restrict the function of infecting DNA). ▪ Mechanism: Restriction enzymes recognize a specific sequence of nucleotides, and produce two types of double- stranded cut in the DNA:BLUNT ENDS and STICKY ENDS.  Sticky ends HindIII EcoRI Blunt ends AluI HaeIII Some of the most commonly used RE 3 types of fragments based on its end. HOWTO USE RESTRICTION ENZYME: ❑ Adding the enzyme to the target DNA in a buffer solution ❑ incubated at 37o C. Enzyme activity unit = Amount of enzyme that will cleave 1 µgr DNA in 1 hour at 37oC. Restricion mapping A technique to determine the relative locations of different recognition sites of different enzymes in a DNA. → cutting DNA with various combination of RE and then determine the sizes of the fragments. e.g From this result, we can conclude thatThe DNA has a single cutting site for each enzyme tested. RESTRICTION MAPPING Isoschizomer & Isocaudomer Isoschizomer Similar recognition site, different restriction site. E g. XmaI 5’-C CCGGG-3’ 3’-GGGCC C-5’ SmaI 5’-CCC GGG-3’ 3’-GGG CCC-5’ Isocaudomer: compatible restriction enzyme Different recognition site, similar restriction site. E g. BamHI 5’-G GATCC-3’ 3’-CCTAG G-5’ BglII 5’-A GATCC-3’ 3’-CCTAG A-5’ DNA Ligase  DNA LIGASE A ‘molecular glue’ Repairs broken phosphodiester bond that may occur randomly or as consequence of DNA replication/recombination. In genetic engineering, used to seal sugar-phosphate chain discontinuities that arise when making a recombinant from different DNA sources. The most frequently used ligase: T4 DNA ligase → from E. coli infected with bacteriophageT4. Most efficient when sealing sticky ends. Optimum in 37oC but usually used at 4-15oC to prevent thermal denaturation of the sticky ends.  DNA Ligase can also join blunt ends DNA fragments with blunt ends generated by different enzymes can be ligated together (with lower efficiency), but usually cannot be re-cut by either original restriction enzyme. -CCCGGG- SmaI -CCC GGG- -AAATTT- DraI -AAA TTT- -CCCTTT- -AAAGGG- Ligations that re-constitute a SmaI or DraI site (CCCGGG or AAATTT) can be re-cut by SmaI or DraI. Mixed ligation products (CCCTTT + AAAGGG) cannot be re- cut by SmaI or DraI.  Principle of cloning Contents Restriction enzyme Host cell Vector Promoter Transforman selection  Plasmids – vehicles for cloning naturally occurring extrachromosomal Ampr DNA molecules. Ori pBR322 circular, double-stranded DNA. 4361bp the means by which antibiotic resistance Tetr is often transferred from one bacteria to another. can be cleaved by restriction enzymes, leaving sticky or blunt ends. LacZ Artificial plasmids can be constructed by MCS linking new DNA fragments to the sticky pUC18 Ori ends of plasmid. Ampr  Cloning Vectors Older cloning vector A cloning vector is a plasmid that can be Ampr modified to carry new genes. Ori pBR322 Plasmids useful as cloning vectors must 4361bp have: Tetr – An origin of replication. – A selectable marker (antibiotic resistance gene, such as ampr and tetr). Newer cloning vector – Multiple cloning site (MCS) (site where LacZ insertion of foreign DNA will not MCS disrupt replication or inactivate pUC18 essential markers). Ori – Easy to purify away from host DNA. Ampr  Chimeric Plasmids Named for mythological beast (chimera) with body parts from several creatures. After cleavage of a plasmid with a restriction enzyme, a foreign DNA fragment can be inserted. Ends of the plasmid/fragment are closed to form a "recombinant plasmid”. Plasmid can replicate when placed in a LacZ suitable bacterial host. MCS pUC18-hCFTR Ori Ampr Host cell If the aim is to isolate a gene for structural analysis → simple host cell If the aim is to express the genetic information in a higher eukaryote→ more specific host cell An ideal host cell should be easy to handle and propagate, should be available as a wide variety of genetically defined strains, and should accept a range of vectors. PROKARYOTIC HOSTS E. coli has been studied in great detail; it is used in many cloning protocols with various strains available for specific application. Other bacterias that are commonly used in cloning procedure: Bacillus sp., Pseudomonas sp., Streptomyces sp. + There is no post-transcriptional modification, i.e. mRNA immediately available for translation — Fewer suitable vector — Difficult to get the recombinant DNA into the cell; need reliable and efficient procedure E. coli, a gram-negative bacteria with a — Lacks organelle and neuronal membrane (some single packed chromosome (nucleoid) which sequence is already known. eukaryotic genes might not function in prokaryot) These problems can be solved by using E. coli as shuttle vectors (perform an initial cloning procedure in E. Coli, isolate required sequence, introduce the purified DNA to target host) EUKARYOTIC HOSTS The most commonly used eukaryotic microbes in genetic engineering is yeast (S. cerevisae) and a range of its mutant cell types. Other possible eukaryotic hosts include fungi (Aspergillus nidulans, Neurospora crassa) and unicellular algae (Chlamydomonas reinhardtii). C. reinhardtii has the advantages of microbes plus the structural and functional organization of plant cells. Other plant or animal cells are usually grown as cell cultures, because it’s easier to manipulate than cells in a whole organism. e.g the most widely used is mammalian cell lines.  Principle of cloning Contents Restriction enzyme Host cell Promoter Vector criteria and types Mobile DNA Transforman selection CRITERIAS Criterias that need to be fulfilled by vector candidates: small DNA molecules → facilitating isolation and handling There must be an origin of replication desirably has selectable marker → for vector detection must have at least one unique recognition site → enabling DNA insertion when forming a recombinant. VECTORS ❑ Plasmid vectors ❑ Bacteriophage vectors ❑ Hybrid plasmid/phage vectors ❑ Vectors for eukaryots ❑ Artificial chromosomes PLASMID VECTORS FOR USE IN E. coli Plasmid = circular, non-essential extrachromosomal genetic materials found in bacteria. Often has antibiotic resistance genes (pDNA) When engineered cells are planted on antibiotic- containing medium, only the plasmid-containing cells survive → convenient selection method PLASMID VECTORS FOR USE IN E. coli Has tra (transfer) and mob Conjugative (mobilising) regions plasmids These regions enable the plasmid to mediate direct conjugation Non- Not self-transmissible, conjugative unable to do conjugation Must have assistance from plasmids conjugative plasmid PLASMID VECTORS e.g pAT153, a deletion derivative of pBR322. 2 fragments from pBR322 are removed using enzyme HaeII → increases copy number threefold. PLASMID VECTORS ▶ another ‘popular’ plasmid family: pUC ▶ usually, pBR1332 and pAT153 are sufficient for simple procedure ✓ pUC has multiple cloning sites (polylinker) which contains several unique restriction sites ✓ Polylinker reside in β-galactosidase gene region (lacZ), coding alpha- peptide. Inserting DNA into polylinker results in non- functional alpha-peptide → direct screening method using chromogenic substrate X-gal PLASMID VECTORS  PLASMID VECTORS Disadvantage of using plasmid: small size of inserted DNA fragment (max 5 kb) ▶ will be problematic when a large number of clone is required e.g. genomic library in which all genomes of an organism must be represented ▶ different vector system is needed → BACTERIOPHAGE BACTERIOPHAGE bacteriophage/phage = the eater of bacteria → bacterial virus P λ H A G E MI3 S BACTERIOPHAGE VECTORS ▶ The genome has been entirely sequenced ▶ has complementary short single stranded region at the end of the linear genome → act as sticky ends ▶ The sticky ends enable circularisation of genome that has infect the cell ▶ the region of the genome generated by the association of both sticky ends after circularisation = cos region BACTERIOPHAGE VECTORS BACTERIOPHAGE VECTORS INSERTION ▶ EcoRI = site to insert DNA VECTORS ▶ In λgt10, EcoRI lies within cI gene (λ repressor) → selection process: plaque formation and morphology ▶ In Charon 16A, EcoRI lies within LacZ → screening using X-gal ▶ Approx. 7-9 kb DNA BACTERIOPHAGE VECTORS ▶ Filamentous phage, shorter than λ ▶ Only infects E. coli that has F-pili (thread- MI3 ▶ like protein appendage, found on conjugation-proficient cells) DNA of the phage that enter the cell becomes double stranded RF (replicative forms) and copied until about 100 ▶ The bacterium is not lysed ▶ single stranded copies eventually produced and extruded from the cell as MI3 particles BACTERIOPHAGE VECTORS Advantages using MI3 phages: the RF is similar to plasmid; the ssDNA produced during infection is useful in DNA sequencing (dideoxy method) ▶ Disadvantages using MI3 phages: ▶ MI3 does not have non-essential genes → manipulation only available on 507 bp intergenic region (usually inserted with polylinker/lacZ sequence for screening purpose) ▶ not efficient when long DNA (>1.5kb) is inserted BACTERIOPHAGE VECTORS Determining the number of bacteriophage present in a suspension: Serial dillution of phage stock with excessive bacteria Plated onto agar and incubated overnight Grown bacteria will form bacterial lawn. phage-infected cell will form clear area (plaques) that can be counted(plaques forming unit).Visible through morphology as well. BACTERIOPHAGE VECTORS Lplaques may formed from lysed bacteria (viral phage) or bacteria with deformed growth (temperate phage) HYBRID PLASMID/PHAGE VECTORS COSMID PHAGEMIDS Replication behavior Like plasmid Either plasmid or phage Packaging/infection Like λ phage Like MI3 phage mechanism Inserted DNA space up to 47 kb up to 42 kb Efficient and specific Has MI3 characteristic Advantage introduction to host, with much greater great cloning capacity capacity Difficult to be processed Must be combined with Disadvantage further other bacteriophage VECTORS FOR EUKARYOTS The aims of genetic engineering in higher (multicellular) eukaryotes can be considered as: To express cloned genes in plants/animals cells in tissue culture for basic research on genetic expression of protein production. Produce a transgenic in which all the cells will carry the genetic modification The well-established vector system for plant hosts is theTi plasmid of Agrobacterium tumefasciens. It may be introduced to host cell by biological entry mechanism. VECTORS FOR ARTIFICIAL CHROMOSOMES Artificial chromosomes simple vectors that mimic the natural construction of chromosomal DNA (telomeres, a centromere, and an ori) in addition to features designed for ease of use, such as selectable markers. Yeast artificial chromosome (YAC) (fig. 5.11) Enable DNA fragments in the megabase range to be cloned (the largest capacity vectors available) The recombinant DNA is maintained essetially as yeast chromosome since it has centromeric and telomeric region There have been some problem of insert instability ARTIFICIAL CHROMOSOMES Bacterial artificial chromosome (BAC) Based on the F plasmid (functional fertility plasmid) that is larger than standard plasmid cloning vector Can accept inserts around 300kb More stable thanYAC Much of the sequencing of the human genome has been accomplished using a library of BAC recombinants  Principle of cloning Contents Restriction enzyme Host cell Vector Promoter Transforman selection PROMOTER The promoter are regions with a specific base sequence, to which RNA polymerase will bind. Promoter acts as the regulator for the level of gene expression (i.e. when, where and how much of the gene product (protein) is produced) by specifying how many mRNA is transcribed for a given gene. Promoter is one of the key features in choosing a vector i.e. Often the aim is to maximise the expressed cloned sequence, hence a vector with highly efficient promoter (strong promoter) is chosen. If the product of the cloned gene is toxic to the cell, weak promoter may be required to avoid cell-death.  PROMOTER Types of promoters used to regulate gene expression: Constitutive promoter Inducible promoter Tissue-specific/development-stage specific promoter Synthetic promoter CONSTITUTIVE PROMOTER These promoters direct expression in virtually all tissues, mostly independent of environmental/developmental factors The first constitutive promoters used for the expression of transgenes in plants were isolated from plant pathogens, e.g opine promoter + High level of production of proteins used to select transgenic cells or plants + Easy protein detection and quantification + High level of expression of transcription factors + Production of compounds that are required during all stages of plant development or requires ubiquitous activity in the plant The search for other constituitive promoters continues... INDUCIBLE PROMOTER the activity of these promoters is induced by the presence or absence of biotic or abiotic factors. A very powerful tool in genetic engineering because it can be easily manipulated to freely turn gene expression on or off in particular developmental stage. Chemically-regulated promoter Physically-regulated promoter regulated by the presence or absence of regulated by the presence or absence of alcohol, tetracycline, steroids, metal light and low or high temperatures and and other compounds. other environmental factors should not be naturally present in the derived from genes from multiple organism where expression of the organisms including bacteria and plants transgene is sought; Include cold- and heat-shock induced Preferably should be derived from promoter, e.g heat-shock system in organisms of distant evolution relative Drosophilla to targeted organism. e.g plant promoters from yeast TISSUE-SPECIFIC PROMOTER The transgenes driven by these type of promoters will only be expressed in tissues where the transgene product is desired, leaving the rest of the tissues in the plant unmodified. More preferable if used in homologous system (same famili, genus, or species); one of the main reasons for the large amount of tissue-specific promoters isolated from particular plants and tissues e.g. Beta amylase gene promoter for seed gene expression tomato pz7 promoter for ovary gene expression tobaco RD2 gene promoter for root gene expression bananaTRX and melon actin promoter for fruit gene expression They may be induced by endogenous and exogenous factors, so they may be also classified as inducible. SYNTHETIC PROMOTER promoters whose parts are synthesized as consensus sequences of the promoter elements found in nature. Consensus sequences = a DNA sequence common to different organisms and having a similar function in each –this includes promoter regions “Promoter elements found in nature” refers to: *Prokaryots have different promoter elements (consensus sequence)  Principle of cloning Contents Restriction enzyme Host cell Promoter Vector Transforman selection Bacterial transformation How to select transformed cells?  Reporter genes Encode protein product that is easily detectable to select transformed cells.  Common reporter genes used Antibiotic resistance gene LacZ gene of E. coli GUS reporter gene Green Fluorescent Protein (GFP) gene from jelly fish Antibiotic resistance gene pBR322 plasmid contains: Ampicillin resistance (β-lactamase gene) Tetracycline resistance Common reporter genes used Antibiotic resistance gene LacZ gene of E. coli GUS reporter gene Green Fluorescent Protein (GFP) gene from jelly fish  LacZ gene of E. coli LacZ gene of E. coli A common reporter in bacteria is the E. coli lacZ gene, which encodes the protein beta-galactosidase. This enzyme causes bacteria expressing the gene to appear blue when grown on a medium that contains the substrate analog X-gal. Common reporter genes used Antibiotic resistance gene LacZ gene of E. coli GUS reporter gene Green Fluorescent Protein (GFP) gene from jelly fish  GUS reporter gene GUS : β-glucuronidase GUS reporter gene is particularly useful in plant molecular biology. It has to be compatible with Agrobacterium- mediated transformation (inserted in binary vector). It is usually used to analyze the activity of a promoter (= the expression of a gene under that promoter). Seedlings stained for PAtFH8:GUS expression in 5-day-old wild-type Arabidopsis (A) and PAtFH8:GUS transgenic plants (B–F). Common reporter genes used Antibiotic resistance gene LacZ gene of E. coli GUS reporter gene Green Fluorescent Protein (GFP) gene from jelly fish  Fluorescent protein GFP: Composed of 238 aa (26.9 kDa) emits bright green fluorescence when exposed to UV Isolated from jellyfish Aequorea victoria. It is usually used to analyze the activity of a promoter (= the expression of a gene under that promoter). YFP: yellow CFP: cyan BFP: blue RFP: red Limpens, Bisseling. 2003. Current Opinion in Biology. 6 (4):343-350 END OF THIS TOPIC References Schleif R. Genetics and Molecular Biology, Second Edition. The Johns Hopkins University Press. Weaver, R.F. 2001. Molecular Biology. McGraw-Hill.