CSB351Y1 Lecture Notes - Lecture 17: Multiple Cloning Site, Insertional Mutagenesis, Genetically Modified Crops
26 May 2018
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Lecture 17: Virus Expression Vectors
Transposable Elements
• Transposon (transposable element) – small piece of DNA inserting itself into another place in genome
• McClintock discovered studying cord – spotaeous reaks i plat’s hroosoes orrelated with color
patterns of corn
• Jumping genes – have ability to move within and between chromosome
• Cut and paste OR copy and paste mechanism
• Can use transposons in biotechnology in future
• Transposons can control gene expression through inactivation of genes via insertional mutagenesis (epigenetic
reprogramming)
• Plant genomes are full of gypsy-like retrotransposons
Advantages of Virus Expression Vectors
• Higher levels of protein expressed over conventional transgenic plants
• Peak protein expression levels can be achieved within short period of time
• Ability to produce proteins which hare potentially toxic to plants at high levels
• Many biosafety concerns associated with transgenic plants can be avoided
Plant Virus Expression Vectors
• Many plant viruses are single stranded (+) sense RNA viruses
• Very difficult to genetically manipulate RNA – 5’ ap ad poly A tail
Construction of cDNA version of genomic RNA of Virus
• Infection cDNA clone
- Start with poly T primers and reverse transcriptase to add dNTPs and produce DNA to RNA template
- Remove RNA template after using RNaseH
- Second strand synthesis with DNA Polymerase I → dscDNA based on original RNA template
- Cut with nuclease S1
- Add terminal transferase (enzyme) and dCTP → make dC tail on ends of our cDNA template
• At multiple cloning site at Pst1 (restriction endonuclease) to linearize plasmid
• Add terminal transferase and dGTPs → poly G tail
• Add virus cDNA and ligase → transform into E.coli select white colonies
Examples of Plant Virus Expression Vectors commonly used today
• TMV can have cDNA gene in front of CP and PVX after CP
- We can add amino acids in and express it on surface of coat protein
- Must take several different clones to make sure to fine one that will be infectious
1. TMV epitope presentation system
- Inserting a short peptide to get epitope presentation system where full virus assembles to have pieces of
short AAs on outside – immune system robust response (good vaccines)
2. TMV polypeptide presentation system
- Make whole polypeptide presentation system
- Get virus particle and also foreign gene produced as protein
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Transposable elements: transposon (transposable element) small piece of dna inserting itself into another place in genome, mcclintock discovered studying cord spo(cid:374)ta(cid:374)eous (cid:271)reaks i(cid:374) pla(cid:374)t"s (cid:272)hro(cid:373)oso(cid:373)es (cid:272)orrelated with color patterns of corn. Plant virus expression vectors: many plant viruses are single stranded (+) sense rna viruses, very difficult to genetically manipulate rna 5" (cid:272)ap a(cid:374)d poly a tail. Construction of cdna version of genomic rna of virus. Start with poly t primers and reverse transcriptase to add dntps and produce dna to rna template. Second strand synthesis with dna polymerase i dscdna based on original rna template. Examples of plant virus expression vectors commonly used today: tmv can have cdna gene in front of cp and pvx after cp. We can add amino acids in and express it on surface of coat protein. Must take several different clones to make sure to fine one that will be infectious: tmv epitope presentation system.
