Lecture 9 (Gateway Cloning) PDF

Gateway ®Recombinational Cloning & Fluorescent Protein Fusion Constructs  2 Intro to weeks 10 lab: protein localization with fluorescent proteins Site-specific recombination: the lambda phage Invitrogen Gateway® recombination cloning t...

Gateway ®Recombinational Cloning & Fluorescent Protein Fusion Constructs  2 Intro to weeks 10 lab: protein localization with fluorescent proteins Site-specific recombination: the lambda phage Invitrogen Gateway® recombination cloning technology ØBP reaction and donor vector ØLR reaction and expression vector ØAdvantages and disadvantages of Gateway cloning  3 Protein localization with fluorescent proteins To further understand gene function, gene knockout studies are often accompanied by gene overexpression, protein localization, protein-protein interaction studies, enzymatic activity, etc. depending on the gene/protein being studied. These techniques often require tagging your protein with peptide sequences (such as epitopes, fluorescent proteins, etc.) at the N- or C- terminus. Protein fusions with fluorescent proteins such as GFP, YFP, etc. (translational fusions, protein-reporter fusions) provide a rapid and simple method for following the subcellular and spatiotemporal localization of a protein. Other techniques, including immunolocalization can be used. To generate these protein-reporter fusions, the coding region of a gene is fused to that of a marker gene, such as GFP, in a vector. The construct is Nuclear localization of Protein-GFP fusion then transformed/transfected into the organism of study. Ø In the lab, you will characterize the localization of CBF4 by generating a CBF4-YFP fusion protein  4 Subcellular localization of CBF4 CBF4 belongs to the AP2/EREBP family of transcription factors, and is closely related to CBF1, CBF2 and CBF3. CBF1/2/3 bind to the promoter region of genes that are regulated by cold stress. While the function of CBF1/2/3 in cold stress response is well characterized, much less is known about the role of CBF4, including its subcellular localization. (Reference reading for CBF4: Haake et al. 2002 http://www.plantphysiol.org/content/130/2/639; required for the lab report + do your own literature search). To understand CBF4 subcellular localization, you will generate a CBF4-YFP fusion protein by recombinational cloning (Gateway® technology, Invitrogen). CBF4 will be cloned in a plant expression vector, which carries the strong 35S promoter and YFP, to generate 35Sp:CBF4-YFP. This construct will be introduced into plants to determine the subcellular localization of the CBF4 protein (in transient assays in N. benthamiana). The same construct can be used to transform Arabidopsis and conduct phenotypic analysis of plants that overexpress CBF4.  5 Experiment Outline attL1 CBF4 attL2 X X Clone CBF4 into a plant expression vector containing YFP 35S attR1 ccdB attR2 YFP by Gateway recombination cloning (LR reaction) LR 35S attB1 CBF4 attB2 YFP Transform LR reaction into E. coli à colony PCR à Plasmid isolation à sequencing (Week 10) Transform plasmid into Agrobacterium Infiltrate plant leaves (N. bentamiana) à transient expression Localize protein by confocal microscopy (Week 11)  6 Site-specific recombination: the lambda phage  7 Site-specific recombination: the lambda phage GATEWAY ® Cloning is based on the site-specific recombination system used by phage lambda to integrate its DNA in the E. coli chromosome. Both organisms have specific recombination (attachment, att) sites: attP in phage and attB in bacteria https://blog.addgene.org/plasmids-101-gateway-cloning From: Thermo Fisher  8 Integration The integration process (lysogeny) is catalyzed by 2 enzymes: the phage l encoded protein Int (Integrase) and the E. coli protein IHF (Integration Host Factor). Upon integration, the recombination between attB and attP sites Int , IHF generates attL and attR sites that flank the integrated phage l DNA (Figure 1). attB and attP sites have a common core sequence of 15bp, flanked by From: Thermo Fisher non-homologous sequences.  9 Excision The process is reversible and the excision is catalyzed by phage l protein Xis (excisionase) and Int, and E. coli IHF. The attL and attR sites surrounding the inserted phage Xis DNA recombine site-specifically Int , IHF during excision to reform the attP site in phage l and the attB site in the E. coli chromosome. From: Thermo Fisher  10 Invitrogen Gateway® recombination cloning technology  11 Gateway® recombinational cloning The GATEWAY® reactions are in vitro versions of the integration and excision reactions. The ultimate goal of the GATEWAY reactions is to make an expression clone, which can be later introduced in the organism of study (by transformation or transfection) to be expressed and characterized. The GATEWAY® cloning is often a two-step process, involving the BP reaction, which generates the entry clone, and the LR reaction, which generates the expression clone. https://blog.addgene.org/plasmids-101-gateway-cloning  12 1. Cloning the gene of interest into a donor vector (many available) using the BP Reaction to generate the entry clone. BP reaction (BP clonase) Smedley and Harwood, 2015 (Methods in Molecular Biology  * ^ ** 13 # ^ ## BP reaction # ** (BP clonase) ^ * ^ ## Figure 1. BP recombination reaction between an attB-PCR product and the pDONR(221 or ZEO) vector to create an entry clone and a by-product. The sequences of the recombination regions may vary slightly depending on the pDONR vector used, but the mechanism of recombination remains the same. Features of the Recombination Region: Shaded regions correspond to those sequences transferred from the attB-PCR product into the entry clone following recombination. Note that the attL sites (#*) are composed of sequences from attB(*) and attP (#), plus the invariant sequence GTACAAA (^). Boxed regions correspond to those sequences transferred from pDONRTM221 or pDONRTM/Zeo into the by-product following recombination. (Invitrogen) Ø Exercise: highlight the similarities and differences in the att2 sites  14 2. Subcloning the gene from the entry clone into a destination vector by the LR Reaction producing the expression clone. LR reaction (LR clonase) Smedley and Harwood, 2015 (Methods in Molecular Biology Many destination vectors are available, once an entry clone has been generated, it can be transferred to many different expression vectors  15 LR reaction (LR clonase) Ø Exercise: highlight the similarities and differences in the att1 sites  16 To make the reactions directional two slightly different and specific sites were developed, att1 and att2 for each recombination site. These sites react very specifically with each other. For instance, in the BP Reaction attB1 only reacts with attP1 resulting in attL1 and attR1, and attB2 only reacts with attP2 giving attL2 and attR2. The reverse reaction, the LR Reaction, shows the same specificity.  17 ?? ØExercise: Look at the attB2 and attP1 sites, could the PCR product be inserted in opposite orientation? I.e could B2 be recombined with P1, instead of P2? ØWhy?  18 ØExercise: Look at the aat1 and att2 sites, could the gene of interest be inserted in opposite orientation?  19 Types of Clonase enzymes Gateway BP Clonase Enzyme Mix Contains both Int (Integrase) from phage and IHF (Integration Host Factor) proteins from E. coli that catalyze the in vitro recombination of PCR products or DNA segments from clones (containing attB sites) and a Donor vector (containing attP sites) to generate Entry clones. Gateway LR Clonase Enzyme Mix Contains a proprietary blend of Int (Integrase), IHF (Integration Host Factor) and Xis (Excisionase) enzymes that catalyze the in vitro recombination between an Entry clone (containing a gene of interest flanked by attL sites) and a Destination vector (containing attR sites) to generate your Expression clone.  20 Summary BP clonase (Int + IHF) LR clonase LR clonase (Int + IHF + Xis) (Int + IHF + Xis) CFP His m m o o r p r p CFP HA m o r p m m o o r p r p GFP Flag m m o o r p r p ? ?  21 1. Generating the CBF4 Entry Clone by Gateway cloning: BP Reaction and Donor vector  1. Generating the CBF4 Entry Clone: BP Reaction 22 Plasmids containing an entry clone can be purchased, if available, or can be generated by cloning a PCR product into pDONR207 Donor vectors through a BP reaction. Gentr To generate the CBF4 entry clone: CBF4 was amplified by PCR using primers with attB1 & attB2 CBF4 sites. The PCR product was then recombined with the donor vector (pDONR207) by the BP reaction using the BP Clonase enzyme. + pDONR207 contains the attP1 & attP2 sites: CBF4 After recombination, CBF4 is flanked by the attL1 and attL2 recombination sites. + Gentr Transform into E. coli DH5α  Guidelines to Design the Forward PCR Primer 23 When designing attB1 Forward Primer, consider the points below (and see Invitrogen guidelines link below). To enable efficient Gateway® cloning, the forward primer MUST contain the following structure: ü Four guanine (GGGG) residues at the 5ʹ end followed by ü The 25 bp attB1 site followed by ü At least 18-25 bp of template- or gene-specific sequences The attB1 site ends with a thymidine (T). If you wish to fuse your PCR product in frame with an N-terminal tag: The primer must include two additional nucleotides to maintain the proper reading frame with the attB1 region (see diagram below). These two nucleotides cannot be AA, AG, or GA, because these additions will create a translation termination codon. Why is this an issue? (25 bp) (18-25 bp) gene GFP https://tools.thermofisher.com/content/sfs/manuals/gatewayman.pdf  Guidelines to Design the Reverse PCR Primer 24 When designing your reverse PCR primer, consider the points below. To enable efficient Gateway® cloning, the reverse primer MUST contain the following structure: ü four guanine residues (GGGG) at the 5ʹ end followed by ü the 25 bp attB2 site followed by ü 18-25 bp of template- or gene-specific sequences 1. If you wish to fuse your PCR product in frame with a C-terminal tag: the primer must include one additional nucleotide to maintain the reading frame with the attB2 region any in-frame stop codons between the attB2 site and your gene of interest must be removed. 2. If you do not wish to fuse your PCR product in frame with a C-terminal tag: your gene of interest or the primer must include a stop codon. Remember that the gene-specific nucleotides need to be in frame with the stop codon. 1. For C-terminal (25 bp) (18-25 bp) Fusions (NO stop codon): gene GFP 2. With STOP codon: gene  Gateway Primers for cloning CBF4 in pDONR207 25 CBF4 was previously cloned into pDONR207 using the following primers (attB1 and attB2 sites are underlined): B1-CBF4-FP 5’- GGGG-ACA AGT TTG TAC AAA AAA GCA GGC TTG-ATG AAT CCA TTT TAC TCT ACA TTC C -3’ CBF4 YFP CBF4-B2-RP 5’- GGGG-AC CAC TTT GTA CAA GAA AGC TGG GTC-CTCGTCAAAACTCCAGAGTG -3’ Note: CBF4 will be C-terminally fused to YFP, to generate CBF4-YFP, hence the CBF4 stop codon was removed. ØFinds the annealing sites of these primers on the CBF4 cDNA sequence (use Clustal Omega)  26 Donor vector and the ccdB gene The donor vector (pDONR207) carries the aacC1 gene (Gentr), which confers resistance to the antibiotic gentamycin. pDONR207 also carries the ccdB gene, which is toxic to E. coli. ccdB makes cloning easier by selecting against vectors that did pDONR207 not take up the insert. Gentr The ccdB gene targets the DNA gyrase , thereby inhibiting growth of most E. coli strains (e.g. DH5α, TOP10). Propagate in DB3.1 E. coli strain Hence, the pDONR207 vector must be propagated in an E. coli strain that is resistant to ccdB, such as DB3.1 strain, which carries the gyrA462 mutation that renders it resistant to the CcdB effects.  27 Recovery of entry clone following BP reaction After the BP reaction, the ccdB gene is released and replaced by the CBF4 gene. pDONR207 The BP reaction is transformed in E. coli laboratory strains Gentr that are sensitive to ccdB (E. coli DH5α). Cells that take up unreacted vectors carrying the ccdB gene CBF4 or by-product molecules retaining the ccdB gene will fail to grow. Ø This allows high-efficiency recovery of the desired clones. + CBF4 + Gentr Transform into E. coli DH5α strain  28 2. Generating the CBF4 expression clone by Gateway cloning: LR reaction and Expression Vector  29 pEarleyGate Destination Vector To generate the expression clone, the gene will be subcloned into a destination Vector. We will Destination use the pEarleyGate101 vector vector (pEARLEY101) The destination vector contains: 1) Promoter for expression in the organism of interest (35S, for strong expression in plants) 2) the ccdB gene, flanked by attR1 and attR2 recombination sites, for negative selection. 3) The polyadenylation and transcription termination sequences (from octopine synthase gene, OCS).  30 pEarleyGate: selectable markers The pEarleyGate 101 destination vector also contains: Destination 1) nptII gene (KanR or Km) which confers vector (pEARLEY101) resistance to the antibiotic kanamycin 2) the cmlA gene (CmR), which confers resistance to the antibiotic chloramphenicol 3) The biapholor resistance gene (BAR), which confers resistance to the herbicide Basta.  Generating a CBF4 Expression Clone: LR reaction 31 The entry clone and destination vector are mixed with pEARLEY101 destination vector the LR Clonase Enzyme. The recombination yields two constructs: the CBF4 Expression Clone and a by-product, the pDONR, which contains CmR + ccdB. CBF4 Entry clone (in pDONR207) The LR reaction is then transformed into E. coli strains + LR Clonase lacking the gyrA462 mutation (such as DH5alpha). E. coli cells that took up the empty destination vector CBF4 (pEarley) and empty donor vector (pDONR) will not be CBF4 expression clone (in pEarleyGate101) able to grow. Only those with the 35Sp:CBF4- + YFP/pEarleyGate101 expression clone will grow. Ø Therefore, all colonies should contain the desired, P1 CmR ccdB P2 recombined construct. (pDONR207) Transform into DH5α strain  32 CBF4 P1 CmR ccdB P2 CBF4/pDONR207 pDONR207 (entry clone) (donor vector) an ain Gent R LR reaction Gent R /K str (LR Clonase) LB 5 α on DH X o AttB1 YFP-rev Pla m int te YFP r R1 CmR ccdB R2 B1 CBF4 B2 YFP sfo R R BA n BA Tra pEarley101 CBF4/pEarley101 (destination vector) (expression clone) Kan R Kan R When DH5alpha cells transformed with the LR reaction are plated on Kan plates, only the cells that took up the expression clone )CBF4 successfully recombined into pEarly101) will grow (bottom, right drawing).  33 AttB1 YFP-rev B1 CBF4 B2 YFP R BA CBF4/pEarley101 (expression clone) Kan R To confirmed successful recombination, you will do colony PCR using AttB1-specific and YFP-specific primers, which will amplify CBF4 cloned into pEarley101 (bottom, right drawing). ØQuestion: Do you think we could use these primers to amplify the ccdB gene in pEarly101?  34 Common E. coli strains used in the lab The majority of all common, commercial lab strains of E. coli used today are descended from two individual isolates, the K-12 strain (such as DH5alpha) and the B strain (such as BL21). Most of the commercial E. coli strains used in molecular biology have a specific purpose: fast growth, high-throughput and routine cloning, cloning unstable DNA, preparing unmethylated DNA, and more. Many mutations that make these features possible are present in most commercial E. coli strains, especially mutations that make major improvements such as those that increase plasmid yield and/or DNA quality. Table 1 from AddGene (https://blog.addgene.org/plasmids-101-common-lab-e-coli-strains) outlines some of the more common genetic changes found in E. coli strains.  35 E. coli strains used in Gateway To propagate empty destination (pEarley) and donor (pDONR) vectors, which have the lethal ccdB gene, you must use an E. coli strain that is resistant to CcdB, such as DB3.1 strain, which carries the gyrA462 mutation. DB3.1 genotype: F´ gyrA462 endA1 glnV44 Δ(sr1-recA) mcrB mrr hsdS20(rB-, mB-) ara14 galK2 lacY1 proA2 rpsL20(Smr) xyl5 Δleu mtl1 Gateway LR reactions are transformed into E. coli strains lacking the gyrA462 mutation, such as DH5alpha strain, so that cells with the empty destination (pEarley) will not be able to grow; only those with the 35Sp:CBF4-YFP/pEarleyGate101 will. DH5-alpha genotype fhuA2 lac(del)U169 phoA glnV44 Φ80' lacZ(del)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 Note: genes listed signify mutant alleles.  36 The CcdA-CcdB toxin-antitoxin system One of the most time-consuming aspects of traditional cloning is the identification of clones that actually contain your insert of interest. ccdB makes cloning easier by selecting against vectors that did not take up your insert. The ccdB gene, located on the F sex factor plasmid of E. coli, is part of a toxin-antitoxin system encoded by the ccd operon, which is responsible for plasmid maintenance during cell division. ccdB codes for the toxic protein (CcdB) that acts as a DNA gyrase poison, locking up DNA gyrase with broken double stranded DNA and ultimately causing cell death. ccdA, another gene found in the ccd operon, codes for the antitoxin protein (CcdA) that protects the cell against the toxic CcdB. Cells that lack ccdA through the loss of the F plasmid, succumb to the toxicity of CcdB. https://blog.addgene.org/plasmids-101-ccdb-the-toxic-key-to-efficient-cloning  37 CcdB resistant E. coli strains While DB3.1 strain has been developed specifically with ccdB-containing vectors in mind, any plasmid that contains the F plasmid (F’ strains) will also be resistant to CcdB, as the native ccdA will be present. Most common cloning strains of E. coli do not contain the F plasmid (and are considered F-) However, there are a few popular lab strains, such as JM109, XL1 Blue or XL10 Gold, that are F’. These strains could possibly be used to propagate and prep your ccdB- containing empty backbones, but should never be used when selecting for recombinant plasmids.  38 Gateway cloning: advantages and disadvantages  39 Gateway cloning: advantages Gateway system allows the cloning of DNA fragments without the need of adding restriction sites, purification of digested vector, etc. (fast, simple and easy procedure). It is also a very versatile system that allows to easily transfer entry clones to different destination vectors, or the cloning of many genes into the same destination vector (high-throughput cloning). Many researchers generated their own Gateway-compatible vector sets for high throughput cloning and expression of genes in plants, bacteria, vertebra embryos and mammalian cell lines. A variety of vector sets also incorporated a range of promoters and terminators, fluorescent protein tags and selectable markers, upstream and downstream of the open reading frames (See the pEarleyGate series).  40 Gateway cloning: disadvantages However, the recombination products are eventually left with “scar” att regions. Such characteristic is undesirable as they add extra amino acids to the expressed protein. Also, cost of the BP and LR clonase enzymes is relatively high compared to conventional restriction-ligation cloning enzymes.  Designing primers for Colony PCR 41 To amplify the cloned DNA fragment by ‘colony PCR’, gene and/or vector specific primers can be used. The template will be the plasmid contained in the colonies, which will be released during the first denaturation step (at 95∘C) of the PCR cycle.  42 Confirming the sequence of the cloned DNA fragment: plasmid sequencing In the end, the most accurate way of confirming the successful cloning of a DNA fragment into a vector is by sequencing. It is important to confirm that: o no mutations have been introduced by the PCR reaction o the gene of interest is cloned in the correct orientation o fusion constructs maintain the correct reading frame, etc. One 35S:CBF4-YFP expression clone will be isolated by using a plasmid DNA isolation kit, which yields pure plasmid DNA isolation. o Sequencing reactions can be inhibited by impurities isolated by regular miniprep methods The purified plasmid will be sent for sequencing. In the lab, you will analyze the sequencing results by looking at the chromatogram.  43 CBF4 In the CBF4-YFP fusion, the C-terminus of CBF4 is fused to the N-terminus of YFP. Ø Therefore, the stop codon of CBF4 has been removed (see sequences of CBF4 FP+RP primers used to generate CBF4 entry clone). 44