Cloning in E. coli (2023/2024) PDF

CLONING IN E. COLI SSCG 2713 – GENETIC ENGINEERING (+LAB) (2023/2024-1) Nurriza Ab Latif (PhD) Department of Biosciences, Faculty of Science, Universiti Teknologi Malaysia BRAIN TEASER  ILLUSTRATE THE GENERAL STEPS IN GENE CLONING WHAT DO YOU KNOW ABOUT P...

CLONING IN E. COLI SSCG 2713 – GENETIC ENGINEERING (+LAB) (2023/2024-1) Nurriza Ab Latif (PhD) Department of Biosciences, Faculty of Science, Universiti Teknologi Malaysia BRAIN TEASER  ILLUSTRATE THE GENERAL STEPS IN GENE CLONING WHAT DO YOU KNOW ABOUT PLASMID?  PLASMIDS Plasmids are molecules of DNA that are found in bacteria, separate from the bacterial chromosome. Plasmids are also referred as “vectors” or “constructs” *also exist naturally in archaea and eukaryotes such as yeast and plants  CHARACTERISTICS OF PLASMIDS Small (a few thousand bp) and widely distributed throughout prokaryotes Usually carry only Does not change in size one or a few genes (constant monomeric unit size). Circular double Stably inherited in extra- stranded chromosomal state Self replicating - Single origin of able to replicate replication independently of the In nature: plasmids often encode chromosome. genes for proteins (e.g. enzymes) that protect the bacterium from one or more antibiotics. Plasmids: Independent genetic elements found in bacterial cells.  In nature, plasmids provide one or more functional benefits to the host such as resistance to antibiotics, degradative functions, and/or virulence. Some phenotypic traits exhibited by plasmid-carried genes Antibiotic resistance Bacteriocin production Antibiotic production Induction of plant tumours Degradation of aromatic Hydrogen sulphide compounds production Haemolysin production Sugar fermentation Enterotoxin production Host-controlled restriction Heavy metal resistance and modification The common features of plasmids Vector element Description Origin of DNA sequence which allows initiation of replication within a plasmid by recruiting transcriptional Replication (ORI) machinery proteins Antibiotic Allows for selection of plasmid-containing bacteria. Resistance Gene Multiple Cloning Short segment of DNA which contains several restriction sites allowing for the easy insertion of Site (MCS) DNA. In expression plasmids, the MCS is often downstream from a promoter Insert Gene, promoter or other DNA fragment cloned into the MCS for further study. Promoter Region Drives transcription of the target gene. Vital component for expression vectors: determines which cell types the gene is expressed in and amount of recombinant protein obtained Selectable Marker The antibiotic resistance gene allows for selection in bacteria. However, many plasmids also have selectable markers for use in other cell types. Primer Binding A short single-stranded DNA sequence used as an initiation point for PCR amplification or Site sequencing. Primers can be exploited for sequence verification of plasmids.  Categories of Plasmids Multiple copies/cell Low copies/cell multiple copies per cell limited number of copies (relaxed plasmids). per cell (stringent Multiple copies of 50 or plasmids). more per cell. One or two per cell. A useful cloning vector needs to be present in the cell in multiple copies so that large quantities of the recombinant DNA molecule can be obtained.  Categories of Plasmids Conjugative Non-conjugative contains a set of transfer does not contain tra genes genes called the tra gene – incapable of initiating which promotes bacterial conjugation. conjugation (the transfer of they can only be plasmids to another transferred with the bacterium). assistance of conjugative generally high molecular plasmids. weight, present as 2-3 high copies. copies per chromosome (low copy number). Example of Conjugation and Mobilization. Conjugative plasmids carry an origin of transfer, oriT, and encode a DNA relaxase (nes) as well as a coupling protein and products for formation of the mating pore (transfer operon). Mobilizable plasmids may carry an oriT that is recognized by the relaxase of the conjugative plasmid (a); encode their own relaxase (mob)/oriT pair that uses the mating pore of a conjugative plasmid (b); or express a replicative relaxase (rep) that recognizes the replication origin (oriV) and is compatible with the conjugative-plasmid coupling protein (c).  Plasmids were the first vectors to be genetically modified and used in cloning. To use them as vectors, they should be modified to contain a limited number of restriction sites and selectable marker genes that detect the presence of plasmid in the host cell. pBR322 M®-T Vector Map and Sequence Reference Points XmnI 1994 ScaI NaeI 1875 2692 T7 ➞ f1 ori 1 start ApaI 14 AatII 20 SphI 26 Amp r BstZI 31 pGEM®-T lac Z NcoI 37 Vector T T SacII 46 (3000bp) SpeI 55 NotI 62 BstZI 62 PstI 73 SalI 75 NdeI 82 SacI 94 ori BstXI 103 0356VA04_3A NsiI 112 126 ➞ SP6 Promega pGEM®-T and pGEM®-T Easy Vector Systems 1. Vector map of yT&A cloning vector Yeastern Biotech yT&A Cloning Vector Kit Protocol PLASMID DNA PURIFICATION Preparation of plasmid DNA from a culture of bacteria follows the same basic steps as purification of total cell DNA. Plasmid and bacterial DNA can be differed in: o Size o Conformation One of the most important steps - lysis of host cells. Effective lysis of bacterial cells is a key step in plasmid isolation as DNA yield and quality depend on the quality of cell lysate used for the purification.  Cells have to be sufficiently broken to permit plasmid out. Incomplete lysis and total dissolution of cells result in reduced recovery of plasmid DNA. Too much contamination from chromosomal DNA must be avoided. Lysis has to be done gently (no vortexing) so that high molecular weight chromosomal DNA can be removed along with cell debris by high speed centrifugation to give cleared lysate.  One traditional plasmid purification technique in use since the 1950s is cesium chloride (CsCl)/ethidium bromide (EtBr) centrifugation (Radloff et al., 1967; Maniatis et al., 1982; and Meselson et al., 1957). To obtain pure plasmid DNA from cleared lysates, isopycnic centrifugation of the cleared lysates in a solution of CsCl (caesium chloride) containing EtBr was done. EtBr binds by intercalating between DNA base pairs therefore causes DNA to unwind.  Isopycnic centrifugation: A technique used to separate molecules on the basis of density. Particle size only affects the rate at which particles move until their density is the same as the surrounding gradient medium. The density of the gradient medium must be greater than the density of the particles to be separated. By this method, the particles will never sediment to the bottom of the tube, no matter how long the centrifugation time. Starting with a uniform mixture of sample and density gradient (A) under centrifugal force, particles move until their density is the same as the surrounding medium (B). CsCl Method by Radloff et al. (1967) Ethidium bromide-caesium chloride density gradient centrifugation CCC DNA molecule (e.g. plasmid) has no free ends and can only unwind to a limited extent, limiting the amount of EtBr bound. Linear DNA molecule (e.g. fragmented chromosomal DNA) has no such topological constraints and can bind more of the EtBr molecules. More EtBr bound, density of DNA- EtBr complex decreases.  More EtBr can be bound to a linear molecule than to a covalent circle, the covalent circle has a higher density at saturating concentrations of EtBr. Thus covalent circles (i.e. plasmids) can be separated from linear chromosomal DNA. Alkaline Lysis Method Most popular method. Employs the use of NaOH at a narrow pH range of 12.0-12.5. The pH range causes denaturation of linear DNA but CCC is not denatured. Production of alkaline lysates involves four basic steps.  Four basic steps in alkaline lysis method Resuspend harvested bacterial cells in Tris·Cl–EDTA buffer containing 01 RNase A. Lyse cells using NaOH/SDS. SDS solubilizes the phospholipid and protein components of the cell 02 membrane, leading to lysis and release of the cell contents. NaOH denatures the chromosomal and plasmid DNA, as well as proteins. The presence of RNase A ensures that liberated cellular RNA is digested during lysis. Neutralize the lysate by adding acidic potassium acetate. The high salt concentration causes potassium dodecyl sulfate (KDS) to 03 precipitate, and denatured proteins, chromosomal DNA, and cellular debris are coprecipitated in insoluble salt-detergent complexes. Plasmid DNA, being circular and covalently closed, renatures correctly and remains in solution. 04 Clear the lysate by either centrifugation or filtration, to precipitate the debris. Purification by Binding Plasmid to A Solid Support Matrix Conditions allowing binding must exist in lysate. Examples of DNA binding supports: o Ion Exchange Resin (low salt) o Diatomaceous Earth (chaotrope, DNA binds in the presence of chaotropic salts) o Silica (DNA binds in the presence of chaotropic salts) Chaotrope: denatures proteins (Guanidine HCl). After binding, wash away impurities using ethanol-based washes. Dry the support. Elution Ion Exchange: Elutes in high salt buffer. Diatomaceous Earth and Silica: Elute in water or low salt buffer. Post elution: o Removal of salt by precipitation with isopropanol. o Concentration of plasmid. PLASMID AS CLONING VECTOR An ideal cloning vector would have the following three desirable properties : SMALL SELECTION UNIQUE SITE Has the ability to confer Single sites (unique sites) Low molecular weight for a large number of REs readily selectable (small in size). phenotypic traits on host preferably in genes with a cells. readily scorable phenotype. Why need antibiotic resistance? Selectable Markers When you transform bacteria, not all cells will receive copies of the plasmid. Selectable marker genes allow for easy identification of transformed cells. The most common selectable markers are antibiotic resistance genes: ampicillin (amp), tetracyclin (tet), chloramphenicol (cam) or kanamycin (kan).  Virtually all plasmids carry resistance to one or more antibiotics. The plasmid makes a protein that destroys the antibiotic (e.g. b-lactamase). Makes any bacteria carrying the plasmid immune to the antibiotic. Ensures that ALL cells in a culture contain the plasmid (look for transformants).  pBR322 DNA is a commonly used plasmid cloning vector in E. coli. Copy number - Medium (10-100) Cloning Sites These are regions of DNA which contain the consensus sequence for numerous REs. Called the multiple cloning site (MCS) or polylinker. The restriction enzymes that cut within the MCS do not cut elsewhere in the vector. This allows for target DNA to be inserted into a specific part of the vector. Directional Cloning Insertion of foreign DNA in a particular orientation. This can be done by making two cleavages with two different REs. Construct foreign DNA with the two restriction enzymes. Foreign DNA can only be inserted in one direction. Screening of Recombinants Once the DNA of interest is inserted to the plasmid at the restriction site in the polylinker, the recombinant plasmid then have to be transformed into bacteria. Within the bacteria these plasmids produce large number of copies through replication. Bacterial cells are plated on media containing: o Appropriate antibiotic o IPTG o X-gal  Recombinant selection with pBR322 ~ Insertional inactivation of an antibiotic resistance gene ~ Cloning into pBR322 in the BamHI site (Insertional Inactivation) Insertional Inactivation in pBR322 2nd selection: cells with plasmids without insertional inactivation of tetR will grow. These 1st selection: are not what we want. only those cells Compare with the with plasmids master plate to retrieve being the clone with insert successfully (that cause insertional transformed into inactivation). them will grow. Cells without plasmid will die.  Recombinant selection with pUC8 ~ insertional inactivation of an antibiotic resistance gene ~  Most modern plasmid vectors therefore make use of a different system. An example is pUC8, which carries the ampicillin resistance gene and a gene called lacZ, which codes for part of the enzyme b-galactosidase. Cloning with pUC8 involves insertional inactivation of the lacZ gene, with recombinants identified because of their inability to synthesize b-galactosidase.  When induced by IPTG, the β-galactosidase (lacZ) gene will be transcribed and the enzyme will use X-gal as a substrate. When X-gal is cleaved, it produces a blue pigment. When there is an insert in the polylinker, lacZ transcription is disturbed, no β-galactosidase is formed and the X-gal not cleaved. Colonies or recombinants with inserts will be white. Transformants without inserts are blue.  pUC18 – a Lac selection plasmid Developed by Joe Messing (Waksman Institute, Rutgers University, USA). pUC18, is a small (2686 bp) plasmid. o This allows it to carry relatively large DNA inserts. In a host cell, it replicates 500 copies per cell, producing many copies of inserted DNA fragments. A large number of restriction sites are available in a polylinker site.  It has a selection system to identify the recombinant plasmids. The identification of recombinant cells can be achieved by a single step process. o By plating onto agar medium containing ampicillin plus X-gal. Cloning using pUC18/19  Identification of recombinants is based on insertional inactivation. lac Z gene -> β-galactosidase intact β-galactosidase produces blue color in presence of X-gal. α-complementation or blue-white screening. Example of IPTG-inducible vector pGEM series (Promega) pGEX series (Pharmacia) pKK223-3 (Pharmacia) pMEX (US Biochemicals) pTrc99A (Pharmacia) pMAL (New England Biolabs) 52 Five (5) steps in cloning a gene (e.g. human gene) in a bacterial plasmid. Step 1. Isolation of vector and gene-source DNA The source DNA comes from any biological sample such as human tissue cells, plant etc. The source of the plasmid is typically E. coli (commercially- available). o Usually carries two useful genes, ampR and lacZ. o The plasmid has a single recognition sequence, within the lacZ gene, for the restriction enzyme used.  Step 2. Insertion of DNA into the vector By digesting both the plasmid and human DNA with the same restriction enzyme we can create thousands of human DNA fragments, one fragment with the gene that we want, and with compatible sticky ends on bacterial plasmids. After mixing, the human DNA fragments and cut plasmids form complementary pairs that are then joined by DNA ligase. This creates a mixture of recombinant DNA molecules. Step 3. Introduction of the cloning vector into cells Bacterial cells take up the recombinant plasmids by transformation. These bacteria are lac Z- unable to hydrolyze lactose. This creates a diverse pool of bacteria, some bacteria that have taken up the desired recombinant plasmid DNA, other bacteria that have taken up other DNA, both recombinant and non-recombinant. Step 4. Cloning of cells (and foreign genes) Transformed bacteria can be plated on a solid nutrient medium containing ampicillin, IPTG, and X-gal. Only bacteria that have the ampicillin-resistance plasmid will grow (transformants) -> blue-white screening. Step 5. Identifying cell clones with the right gene In the final step, the thousands of bacterial colonies with foreign DNA can be sorted out to find those containing our gene of interest. Nucleic acid hybridization, which depends on base pairing between our gene and a complementary sequence (i.e. a nucleic acid probe) can be used. The sequence of our RNA or DNA probe depends on knowledge of at least part of the sequence of our gene. A radioactive or fluorescent tag labels the probe.  The probe will form hydrogen-bond specifically to complementary single strands of the desired gene. After denaturation (separating) the DNA strands in the plasmid, the probe will bind with its complementary sequence, tagging colonies with the targeted gene.  One disadvantage in plasmid vectors is it can carry up only to 10 kb of inserted DNA. When larger DNA fragments are required, genetically modified strains of lambda phage are used as vectors.  PLASMID AS EXPRESSION VECTOR Criteria of expression vectors: Multicopy plasmids - gene dosage. Multiple selective (resistance) markers - Strong and different Ampicillin, Tetracycline, promoters - lac, trp, trc, Kanamycin, etc. tac, temperature- Regulatable promoter - inducible. tightly regulated promoter. Additional criteria: Multiple promoters (shuttle vectors for different hosts). Multiple ori sequences (e.g. bacterial and animal ori, bacterial and plant ori). Production of fusion proteins. Easy purification elements e.g. N-terminal tags (e.g. polyHis) with cleavage site. Incorporates translation signals. Utilize protease defective hosts (e.g. E. coli BL21(DE3), BL21(DE3)pLysS). Usage of Expression Vectors Used for the deliberate expression of target sequences in a heterologous host, ○ protein expression in E. coli ○ protein expression in fungal, insect or mammalian cells Purpose: ○ to produce large amounts of a particular protein for biochemical or structural analysis ○ to examine the function of a protein Outcome of Uncontrolled Expression Expression often deleteriously affects growth of the host cell (hazardous, bad effect on host). Expression is usually tightly regulated using specific promoter constructs. Expression can be divided into two main phases: i. Cell growth phase (biomass generation) - expression switched off ii. Expression phase - expression induced Expression in pET vectors  Expression in pET vectors Expressed only if IPTG is added Further reduce transcription of cloned gene Bind to residual T7 RNA polymerase in the absence of induction and inactivate it Common Expression Machinery Fusion Proteins Increase stability. Easy detection/assay o Can utilize spectrophotometry. o Can employ binding assays. o Can employ antibody screening. May contain export signals to allow transportation of proteins into the periplasm or to outside of the cell. Allow affinity purification Glutathione-S- transferase Vectors designed for protein recovery: Maltose Binding Protein with Amylose Column Cloning in pMAL vector generates fusion protein with MBP, with protease target site in fusion linkage. Cytoplasmic expression. Protein recovered using amylose affinity column. Fusion protein cleaved using Factor Xa protease. Recombinant protein recovered by affinity chromatography Vectors designed for protein recovery: His Tags His6 Tag o add six consecutive His to either end o binds metals Addition of a few residues should have minimal effect on recombinant protein Purification of recombinant fusion proteins (with intein) expressed in E. coli SPECIALIZED VECTORS Vectors designed for specific applications, e.g. protein expression, gene expression, and cellular analysis. Examples of specialized vectors: expression vectors, shuttle vectors. Expression Vector Expression vectors have been developed for both prokaryotic and eukaryotic hosts. Many cloned genes are not expressed efficiently in a new host. pET is an example of a specialized vector: contain genes that will increase the level of transcription of the cloned gene and make its transcription subject to specific regulation. Signals to improve the efficiency of translation may also be present in the expression vector. Shuttle Vector: pYES2 A shuttle vector is a cloning vector that can stably replicate in two different organisms. Shuttle vectors allow cloned DNA to be moved between unrelated organisms (e.g., from one bacterium to another). E. coli replication origin and selectable marker eukaryotic replication origin, selectable marker promoters/enhancers, polyA signals. Shuttle Vector: pSE420 developed by Invitrogen Corp. polylinker – multiple cloning site. trc promoter (trp+lac) – a strong hybrid promoter upstream of lac O (operator) and lambda PL promoter. S/D – Shine-Dalgarno sequence – expressed on the resulting mRNA for efficient translation. T1 and T2 – two transcriptional terminators- prevents transcription of the entire vector. lac I – lac repressor which regulates transcription. pSE420 Shuttle Vector: pBC KS+ pUC ori (high copy number) CAM (chloramphenicol) resistance polylinker blue/white screen for inserts (lac Z’) can produce ssDNA (f1 ori) T7/T3 promoters to make strand-specific probes pUC origin 1158–1825 MCS chloramphenicol resistance ORF 2298–2849 Kpn I chloramphenicol f1 (+) ori f1 (+) origin 135–441 pBC KS+ β-galactosidase α-fragment 460–816 3.4 kb P lac multiple cloning site 653–760 lacZ' lac promoter 817–938 Sac I pUC origin 1158–1825 MCS chloramphenicol resistance ORF 2298–2849 Kpn I chloramphenicol pBC KS+ pUC ori 3.4 kb P lac pBC KS (+/–) Multiple Cloning Site Region (sequence shown 598–826) Not I BssH II T7 Promoter Sac I BstX I Sac II Eag I Xba I pUC ori TTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGA... M13 –20 primer binding site T7 primer binding site SK primer binding site... Hinc II Apa I Bsp106 I Acc I EcoO109 I Spe I BamH I Sma I Pst I EcoR I EcoR V Hind III Cla I Sal I Xho I Dra II Kpn I...ACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACC......SK primer binding site KS primer binding site pBC KS (+/–) Multiple Cloning Site Region T3 Promoter BssH II β-gal α-fragment (sequence shown 598–826)...CAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCC Not I T3 primer binding site BssH II T7 Promoter M13 Reverse primer binding site Sac I BstX I Sac II Eag I Xba I TTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGA... M13 –20 primer binding site T7 primer binding site SK primer binding site... Bsp106 I Hinc II Acc I http://www.genomex.com/vector_maps/pBC_KS_plus_map.pdf Apa I EcoO109 I TRANSFORMATION  Uptake of DNA from outside of cell. Uptake and stable incorporation of naked DNA by bacteria. E. coli not naturally transformable. o Have to undergo some form of physical and/or chemical treatment that enhances their ability to take up DNA. Competent Chemically competent cells Chemically competent cells are cells that were made competent with a salt treatment followed by a heat-shock step. This process permeabilizes the cell membrane, allowing plasmid entry. Protocols using CaCl2 or MgCl2 are the most common method for making chemically competent cells, but some protocols involve other salts or combinations of various salts and chemicals. List of salts and chemicals used to make chemically competent cells CaCl2: Neutralizes the negative charges of the phospholipid bilayer and DNA. DMSO: DMSO gathers at the hydrophilic heads of the lipid bilayer, weakening the forces. As DMSO concentration increases, the thickness of the bilayer decreases, increasing membrane permeability. MgCl2: Works the same way as CaCl2, but allows better DNA binding to the cell. PEG: Shields the negative charges of the phospholipid bilayer and DNA. RbCl: Works the same way as CaCl2 and MgCl2. Some researchers prefer RbCl when higher competency is required. The salt (chemical) treatment neutralizes the negative charges of the phosphate heads and the negatively charged DNA. Neutralizing these charges eliminates the natural repulsion, allowing DNA to move closer to the cell. Transformation Methods: CaCl2 Perturb the membrane using chaotropic agents-chemicals. Small proportion of cells become competent. 1:10,000 DNA molecules taken up. 1:100,000 linear DNA molecules taken up. Plasmids larger than 10 kb taken up poorly. Principles of CaCl2 Transformation Discuss the rationale of the following steps: o Cells incubation on ice o Heat shock o Nutrient Broth incubation Principles of CaCl2 Transformation Transformation Methods: Electroporation Bacterial cell is exposed to high intensity electric field pulses, which destabilise the membrane. The destabilised membrane is rendered permeable to exogenous molecules present in the media. Good alternative for bacteria that cannot be transformed using chaotropic agents.  For in vitro electroporation, a suspension of target cells is mixed in a conductive solution with the molecule you wish to introduce into the cells and placed in a cuvette. Electroporation cuvettes have metal plates on either side of the sample chamber that allow an electrical current to be passed through the mixture. The cuvette is placed into a chamber of the electroporator that has corresponding electrical contacts, enabling the cuvette to form an electrical circuit. Controls on the electroporation machine allow the user to set the voltage, waveform and duration of the electrical pulse to be delivered, which should be optimized to the target cell type. An electrical pulse is then passed through the sample chamber. Schematic diagram showing disruption of the cell membrane and pore formation during electroporation. Schematic diagram showing the steps and associated charges during electroporation that lead to the introduction of exogenous material into the cell, in this case a plasmid. What to consider before bacterial electroporation? 1. Healthy culture of cells. For bacterial strains, mid-exponential phase cultures that are actively dividing are a good rule of thumb. 2. Cells need to be free of salts as otherwise the current will pass around them in the solution when the electrical pulse is delivered. Therefore, wash the cells to remove salts; ice cold 0.5 M sucrose can be a good option. 3. Always place the cuvette and substance to be introduced on ice to keep things as cold as possible. 4. Pipette the washed cells into the cuvette and gently add the substance to be introduced, pipetting gently to mix. 5. Before placing the cuvette in the electroporator, ensure it is thoroughly dried. 6. Deliver the electrical pulse and check the time constant is within the expected parameters. 7. Add ice cold media to the cells and allow them to warm back up to their normal growth temperature in the incubator. Try to prevent sudden shocks as this can increase cell death and reduce the efficiency of your experiment. 8. Once they are warm and actively dividing again (which will depend on the cell type or strain being cultured), cells can be cultured as normal.