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39 Cards in this Set
- Front
- Back
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Plant biology frontiers
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agriculture and GM
Production of high value LMW compounds protein production (therapeutic or industrial) |
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Advantages of using plants as biological systems
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- simpler growth requirements
- easier to store genetic material (as seed), no need to continuously grow -glycosylation of proteins resembles animal much closer -no risk of virus contamination as there are no common viruses -readily transformed genetically (most crops) - transient transfection systems => allow rapid scale-up production |
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Disadvantages
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-stable transformation occurs via illegitimate recombination mechanisms => random integration can cause problems
- cost advantages are limited if plants need to be in containment facilities - growth is slower than microbial growth, but also more persistent - higher persistence means higher risk if genetically modified plat material escapes in the environment |
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main methods of transformation
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Agrobacterium & Biolistic
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Agrobacterium
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nuclear transformation
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Biollistic
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plastidic transformation
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Agrobacterium evolution
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plant pathogen--> plant parasite
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Agrobacterium Transformation
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-transfers DNA to plant/yeast/ human cells
- is the causative agent to crown gall, (favourable environment for bacterial growth - Tumour formation requires Ti plasmid (180-270kb) - part of the Ti plasmid is transferred to the nucleus (T-DNA) - T-DNA encodes genes for auxin and citokinin biosynthesis - also encodes genes required for opine and agropine production, which are transcribed by the plant (have plant transcription factor binding sites and poly-adenylation signals) which only the bacterium can utilise |
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Ti plasmid
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stands for tumour inducing
T-DNA element : -the part of the plasmid that gets transferred to the plant - is flanked by two 25-bp direct repeats (left and right T-DNA borders) - Vir protein machinery becomes induced by specific host signals => Vir1 and Vir2 nick both borders at the bottom strand of the T-DNA molecule => ss T-DNA (T-strand) => becomes exported together with other proteins into the host cell (through a channel formed by VirD4 and VirB proteins) => T-strand forms T-complex (VirD2 attached to 5' of T-strand and assembles with many VirE2 molecules => complex imported into host cell nucleus T-complex associates with host factors such as AtKAPalpha Nuclear Importin |
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pros of using Ti plasmid
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- only 24bp border repeats flank the T-DNA required
- vir genes required act in trans - T-DNA transfer/integration don't depend on oncogenes |
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strategy
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separate transferred genes from the Vir genes required for the transfer into separate plasmids
1) T-DNA plasmid: replicates in Ecoli/ Agrobacterium - has selectable markers (bactrial/plant hosts) - left/ right borders - lack oncogenes 2) Disarmed Ti plasmid: contains genes (vir genes) required for transfer of T-DNA plasmid (replace oncogenes with genes of interest) |
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Agrobacterium mediated transformation
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1. make construct
2. transform argobacterium 3. transfer T-DNA into plant |
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issues with Argobacterium mediated transformation
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-inefficient for monocots
- tissue culture required (chimera risk) - random insertion site - transgene containment |
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Biolistics and plastid transformation
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???? finish lecture
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plastid transformation
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extremely high copy numbers
large numbers of plastids large number of genomes/ plastid => high number of molecules +25% of total protein expressed in leaf protein + no gene silencing + good containment + polycistronic cinstructs - re-engineer genes to optimize codon usage for prokaryotic environment |
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construct has
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for single gene:
promoter, marker gene, terminator, promoter gene of interest, terminator polycistronic RBS separating ORFs selection: use metabolite (high transformation efficiency) => toxic to untransformed cells antibiotic selection (lower) |
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plastid genome
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follows prokaryotic rules in terms of replication and structure
contains several small and one large inverted repeat regions. genes are duplicated, one is dispensible, can integrate through homologous recombination (single copy is adequate) must perform two rounds of selection ensure all plastids contain construct other advantage: maternal plasid inheritance (those in pollen degrade) => no spread of transformed plastids in the field!! |
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plastid versus nuclear transformation
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EXAM PREP WHAT?
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Vectors
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bacterial replication sequence
bacterial selection marker 5kb homology part separate resistance marker from cargo co bombard probability of cotransformation is higher plant promoter (more yeast like) 500 bp from start codon |
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Classical
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semi random integration of
within/ between gene integration T-DNA has preferance for such integration D1,2 proteins interact with host proteins basic host transcription factors, which are in close proximity with genes=> causes the bias in integration site integration mediated by host non-homologous end joining need for targeted insertions |
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s
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misexpression due to random integration
=> yield loss? => need to control UG 99 rust leads to diminished growth in weat to introgress resistance to all these is impossible targetted gene replacement => GMOs production becomes easier no need for selection marker (antibiotic resistance) which is not well approved. -> deliver right allele -> |
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Non-homologous Repair mechanisms
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gene disruption
(remove segment of gene) or generate an insertion - make cut - provide in huge number molecules with fragment, that might be ligated within two DS breaks can lead to inversion/ deletion |
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Homology-directed repair
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homology flanks => integration
can change gene allele -> perfect method for |
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DSB repair
1) NHEJ 2) HR |
NHEJ
Ku70 / Ku80 sense DSB and position at ends ligase 4 anneals those together * lossed DNA sequences due to trimming of ends at staggered breaks => only an emergency repair mechanism (survival of the cell) Homology dependent DSB attracts MRN complex=> bind to ends =? recruit kinase ATM => crossphosphorylated ATMs activate expression of homology dependent DNA repair ( in humans activates guardian of the genome P53 (master transcritpional regulator which activates repair process/ orchestrates cell death eetc etc ) => single stranded ressection? => ss ends strands loaded with rad51 => holiday junction formation => invasion of sister chromatid (provides the information for repair) ===> prevalent in mosses (can do the same gene manipulations as in Yeast) |
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In higher plants there are more NHEJ mechanisms
than HR => competition => most DSB are repaired by NHEJ |
higher plants can survive this due to polyploid => after differentiation => 64 - 120 copy number of genome
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Steps in genome editing
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first -> introduce DSB on target
second -> provide template third -> enhance homology dependent repair. use NHEJ mutants? |
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replacement of alelle B by allele A
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s
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TALENS
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two aa make specific contacts => structure
Natural TALs have several repeats => recognize specific sequences addition of nuclease domain of FokI (ss break inducing) |
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Zinc fingers
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recognises 3 bases, slightly less specific
not a problem |
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comparisson
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zinc fingers
edge effects, neighbouring fingers influence each other validation of |TALENS is more straightforward, more difficult to clone repeats off-target effects still observed (critical for human gene therapy, can be fatal) |
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assay for validation
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in vitro digestion assay
->target plasmid. add protein. check if it cuts T-DNA repair assay - nuclease and target on the same molecule Transgene repair assay -would it happen in trans Whole plant assay - everything transformed into the plant target molecule in plant. agrobacteria infiltrated QQR+ target |
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ZFNs and TALENs
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-chimeric nucelases,
(programmable sequence-specific DNA binding modules linked to a DNA cleavage domain) - stimulate NHEJ or HR at specific genomic locations - |
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ZFNs
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~30 aa per finger, conserved betabetaalpha config.
- aa. on the surface of the a-helix contact 3bp in the major groove of the DNA - modular structure allows design of custom DNA binding proteins - discovery of linker sequence allowed the construction of unnatural arrays of ZFN that recognise 9-18bp in length -. s[pecificity 1:68 billion |
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TALEs
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naturaly occuring in Xanthomonas
-DNA-binding domains (33-35 aa repeat domains - each recognises a single base pair - specificity determined by two hypervariable aas (repeat variable di-reidues, RVDs) - long arrays of TALEs can be made (without re-engineering of the linker sequence necessary) to target single sites in the genome + greater design flexibilty (single base recognition) - difficult to clone repeat TALE arrays (extensive identical repeat sequences) => there are technologies that overcome that issue (Golden gate molecular cloning) - targetting limitatioon: need to start with a T base |
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genome editing
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traditionally achieved by homologous recombination (add fragment flanked with homologous regions-> homologous recombination)
-> frequency of HDR rises dramatically by introducing DSB - targeted nucleases become useful: co -deliver a site-specific nuclease with a donor plasmid bearing locus specific homology arms-> integrate single/multiple transgenes - NHEJ creates small deletions-> frameshift mutations-> generation of null phenotypes |
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CRISPR/Cas
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clustered regulatory interspaced short palindromic repeat
bacterial intergenerational acquired immunity against invading DNA via RNA-guided DNA cleavage - short segments of foreign DNA (spacers) integrated within the CRISPR locus -> transcribed + processed into CRISPR RNA (crRNA) -> crRNA anneal to transactivating crRNAs (tracrRNAs)=> direct sequence specific cleavage of pathogenic DNA by Cas proteins *Cas9 requires a 'seed' sequence within the crRNA and a conserved dinucelotide-containing protospacer adjacent motif (PAM) => can therefore be retargeted to cleave any DNA sequence by redisigning the crRNA two separate RNA molecules originally => can be fused to provide a functional sinthertic chimera (single-guide RNA) depending on Cas enzyme, downstream needs to be a PAM how to deliver both molecules? -> express Cas under RNApol II dependent promoter (control) sgRNA expressed under RNApolIII (specific transcription start point, only one single base requirement g/a |
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sgRNAs
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s
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plant factories advantages
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speed- transient expression systems
cost;= production/ purification costs reduced scalling: no large capital outlay required safety: free from endotoci/ pathogens ethical: no animals/ animal products needed low-tech: easier to deploy in developing countries, avoids long distance transport of vaccines 9continous cold chain |
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plant factories disadvantages
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GMO - suspicion of public (overcome by using transient/ chloroplast based expression)=> containment
cost: containment costs novel: conservative large industries (no experience with plant technologies) |
