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Lecture 9 Recombinant DNA, Vector Design and Construction
Neal Stewart and Dave Mann
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Discussion questions 1. What basic elements should be included in the design and construction of an efficient ubiquitous and constitutive plant gene expression vector? 2. Discuss the advantages and disadvantages of recombination cloning technologies versus traditional restriction digestion and ligation technology. 3. Describe a novel strategy to generate a T-DNA vector that allows the expression of several genes from a single position in the genome. 4. Discuss the advantages and disadvantages of using plastid vectors for plant transformation and gene expression. 5. Describe ways in which transgene technology could be made more acceptable to the public.
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Nucleotide base pairing
A’s pair with T’s G’s pair with C’s Nucleotide base pairing occurs through “hydrogen bonding” Strands have directionality from 5’ to 3’ and when paired strands are in “antiparallel” orientation
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Figure 8.1
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Transgenic plants-Agrobacterium
Any gene, any organism The new plant will pass the transgene to its progeny through seed.
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Biolistics Consumer reports, Sept. 1999
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Steps to make transgenic plants—lotta transformation
Make transformation cloning plasmid vector Transform bacteria (usually Escherichia coli) to maintain clone Characterize plasmid (restriction digest and sequencing) Transform Agrobacterium (if using Agrobacterium) and characterize Transform plant
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Recombinant DNA history
1966 The genetic code is deciphered when biochemical analysis reveals which codons determine which amino acids. 1970 Hamilton Smith, at Johns Hopkins Medical School, isolates the first restriction enzyme, an enzyme that cuts DNA at a very specific nucleotide sequence. Over the next few years, several more restriction enzymes will be isolated. 1972 Stanley Cohen and Herbert Boyer combine their efforts to create recombinant DNA. This technology will be the beginning of the biotechnology industry. 1976 Herbert Boyer cofounds Genentech, the first firm founded in the United States to apply recombinant DNA technology 1978 Somatostatin, which regulates human growth hormones, is the first human protein made using recombinant technology. 1983 Kary Mullis does PCR Kary Mullis publishes method. Patents follow. 2000 Gateway cloning
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Key enzymes Restriction endonuclease DNA ligase Taq DNA polymerase
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Cloning platforms Restriction enzymes/ligase PCR-based methods
Gateway and other site specific recombination methods Golden Gate and other “parts-based” modular construction methods
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Restriction enzyme-ligation
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Figure 8.3
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Table 8.1 Restriction Endonucleases
Enzyme Source Recognition sequence Cut Ends EcoRI Escherichia coli RY13 GAATTC G AATTC 5overhangs CTTAAC CTTAA BamHI Bacillus amyloliquefaciens GGATCC GATCC CCTAGG CCTAG HindIII Haemophilus inflenzae AAGCTT A AGCTT TTCGAA TTCGA KpnI Klebsiella pneumoniae GGTACC GGTAC C 3overhangs CCATGG CATGG NotI Nocardia otitidis GCGGCCGC GC CGCCGG CGCCGGCG GGCCGC CG PstI Providencia stuartii CTGCAG CTGCA GACGTC ACGTC SmaI Serratia marcescens CCCGGG CCC GGG Blunt ends GGGCCC SacI Streptomyces achromogenes GAGCTC GAGCT CTCGAG TCGAG SstI Streptomyces stanford TaqI Thermophilus aquaticus TCGA T CGA AGCT AGC XbaI Xanthomonas campestris TCTAGA CTAGA AGATCT AGATC
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Figure 8.2
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Figure 8.4
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Figure 8.5
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Transformation vector requirements
Origin of replication Bacterial selectable marker Gene constructs of interest T-DNA borders and other Agrobacterium genes if using Agrobacterium Compatible with helper plasmid if using Agrobacterium
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Figure 8.6 Figure 8.6 The Ti plasmid of Agrobacterium tumefaciens showing the origin of replication, the region encoding the virulence (vir) genes, and the transfer-DNA (T-DNA). The T-DNA is flanked by 25-bp direct repeats, known as the left and right border sequences (LB and RB, respectively). The vir genes are required for T-DNA processing and transfer to the plant cell. The T-DNA is stably integrated into the nuclear genome of the plant cell, and genes encoded within it, necessary for the biosynthesis of the plant growth hormones, cytokinin and auxin, result in the formation of the characteristic tumorous growth associated with crown gall disease. The T-DNA also encodes opines (nopaline and octapine) that provide the Agrobacterium with an exclusive nitrogen source. This provides Agrobacterium carrying the Ti plasmid with a competitive advantage over Agrobacterium that do not.
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TABLE 8.2 Commonly Used Bacterial Selectable Marker Genes
Antibiotic Antibiotic Resistance Gene Gene Source Organism Streptomycin/ Spectinomycin Aminoglycoside adenyl transferase gene aadA E. coli Kanamycin Neomycin phosphotransferase gene nptII (neo) E. coli Tn5 Chloramphenicol Chloramphenicol acetyltransferase gene cat Ampicillin -Lactamase bla E. coli Tn3 Tetracycline Tetracycline/H+antiporter tet E. coli Tn10
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Typical components of transformation vector: making a construct
Selectable marker cassette (with promoter and terminator) Gene of interest cassette (with promoter and terminator) Scorable marker cassette (with promoter and terminator) What happens if the promoter is missing? Is there ever a time when a promoterless construct is desirable?
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Figure 8.7
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Figure 8.8 Figure 8.8 A generic plant binary vector with two origins of replication, the pVS1 ori for propagation in Agrobacterium and the ColE1 ori for propagation in Escherichia coli. The backbone of the vector contains an antibiotic resistance gene for bacterial selection (kanamycin resistance), and the T-DNA contains a plant selectable marker and the gene of interest (GOI).
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Figure 8.9
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PCR videos! How PCR works: http://youtube.com/watch?v=_YgXcJ4n-kQ
PCR song!
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What is cloning?
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http://upload. wikimedia. org/wikipedia/commons/thumb/6/66/Scissors
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So, you’ve cut out a gene… Now what?
Gene for blue flowers Digest vector DNA with restriction enzyme How did you amplify this gene? Plasmid vector What are essential components of vector DNAs? Ligate gene into vector Extract plasmid DNA Transform plant
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Solving problems of insert orientation
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Problems with conventional cloning
Inconvenient restriction sites Vector construction is laborious Time-consuming reactions
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Blunt-end Ligation G CTTAA EcoRI ACGTC G PstI G C EcoRI C G PstI C G G
Klenow Fragment C G G C Ligase
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Site-specific Recombination: Gateway™ Cloning
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Bacteriophage λ
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Bacteriophage λ
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Figure 8.11
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Gateway™ cloning Figure 8.12
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Figure 8.13
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Transform into DH5α E. coli cells
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Transform into DH5α E. coli cells
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Functional analysis of open reading frame (ORFs)
Why do we need so many types of vectors? What are some different applications in plants? Functional analysis of open reading frame (ORFs) Overexpression and knockdown (RNAi) of specific genes. Multigenic traits for crop improvement Analysis of the expression level/specificity/ inducibility of promoters
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RNA interference pathway
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Figure 8.14 Figure 8.14 Plant gene expression vectors for conventional cloning using restriction digestion and ligation (a) and Gateway® recombination cloning (b). The first vectors shown in (a) and (b) are designed to allow a gene to be ectopically expressed in a plant cell. The second vectors shown for each category contain the GFP (green fluorescent protein) gene. These vectors are designed to effect protein fusions with GFP to help identify the subcellular target of a protein under investigation. Ideally, three vectors for each type are frequently made, one for each reading frame, to ensure that a perfect fusion between the GOI and the marker gene is made. The insert DNA must be in an “open” ORF configuration (described in the text) so that no stop codon is present between the GOI and the marker gene.
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Figure 8.15 Figure 8.15 Gene silencing in plants can be achieved using inverted repeat transgene constructs that encode a hairpin RNA (hpRNA). Using Gateway® cloning technology, the production of such inverted-repeat transgene constructs can be achieved efficiently, since DNA fragment orientation during the excision and integration process is maintained and the Gateway® recombination cassettes are arranged in opposite orientations with respect to each other.
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Figure 8.16 Figure 8.16 Multisite Gateway® allows several DNA fragments to be cloned within a single vector construct. More recent advances in the design of new att recombination sites have permitted the assembly of up to five DNA molecules within a single vector construct, but none have been designed as yet for plant transformation
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Gebert et al., The Plant Cell, Vol. 21: 4018–4030, December 2009
Multiple promoters from the MRS2/MGT gene family fused to the GUS gene and expressed in Arabidopsis thaliana Gebert et al., The Plant Cell, Vol. 21: 4018–4030, December 2009
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Figure 8.17 Figure 8.17 Excision of selectable marker gene following T-DNA insertion into the plant genome. XVE is a chimeric transcription factor. It contains three functional domains, a LexA DNA binding domain (X), the VP16 activation domain (V), and the estrogen receptor binding domain (E). The G10-90 promoter drives the constitutive and ubiquitous expression of XVE in transformed plant cells. The XVE protein is then bound as a monomer in the cytosol of the cell by a chaperone protein HSP90, and the target gene is transcriptionally inactive. Application of -estradiol causes a conformational change in E, which leads to the release of HSP90 and dimerization of the receptor. On dimerization, the receptor is activated, allowing the protein to translocate to the nucleus of the cell where it binds OLexA binding sites of the promoter that is placed upstream of the Cre recombinase. The VP16 activation domain activates RNA polymerase II, leading to the transcription of the Cre recombinase gene. Cre recombinase allows recombination to occur between the LoxP sites removing all intervening genes, including the selectable marker gene.
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Figure 8.18 Figure 8.18 Site-specific integration is achieved by two homologous recombination events, one on either side of the DNA fragment to be integrated. During insertion, the targeted region of the vector replaces the targeted region of the plastid genome, and the vector backbone is lost. The inserted DNA fragment contains a selectable marker (here, the aadA gene encoding aminoglycoside 3-adenylyltransferase, providing spectinomycin resistance) and can contain either a single gene flanked by independent 5 and 3 regulatory regions, including a promoter; a 5 UTR and a 3 UTR; or, as is the case here, multiple genes with a single promoter that regulates the expression of the operon with individual ribosome binding sites (RBS), upstream of each ORF. In this example, the Bacillus thuringiensis (Bt) cry2Aa2 operon is inserted in the plastid genome generating insecticidal proteins in plant cells. The 16S ribosomal RNA (rRNA) promoter (Prrn) drives the expression of the aadA gene and the three genes of the cry2Aa2 operon. The terminator is the psbA 3 region of a gene encoding the photosystem II reaction center component of the tobacco chloroplast genome.
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Vectors derived from plant sequences
Public acceptance of GMOs linked to concerns of the origin of DNA employed Ironically, wild-type plant cells already contain bacterially-derived genomes T-DNA could be replaced with P-DNA Replace viral promoters with plant promoters
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Figure 7.20
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Pollen-specific promoter LAT52 activates recombinase in tobacco pollen
excision LB RB Pollen genome LAT52 FLP Recombinase NOS GUS:NPTII 35S p loxP-FRT FRT loxP Luo et al Plant Biotechnology Journal 5(2): 263 - 274. (Courtesy of Moon’s poster) A B
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Example of a plant expression vector set
pANIC Made for switchgrass transformation: BioEnergy Science Center (BESC)
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RNAi of lignin biosynthetic genes in switchgrass
The pANIC vector set Functional in switchgrass and rice Overexpression (OE) and suppression (RNAi) of genes ZmUbi1 CaMV 35S Protein tag for OE – AcV5 RNAi of lignin biosynthetic genes in switchgrass Shen & Dixon, Noble Foundation 53 Mann et al. Plant Biotechnology Journal 2012 53 53
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pANIC - Reporter cassette
GUS staining photos courtesy of Zach King PvUbi1 promoter Histochemical GUSPlus Fluorescent pporRFP – novel RFP Comparable to DsRed Ex/Em = 578 nm/595 nm Brightfield – 5ms RFP – 2s GFP – 10s DsRed pporRFP 54 54
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pANIC Vectors:
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Modular “parts” based cloning
Golden Gate is one method
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