Engineering the plastid
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Transcript of Engineering the plastid
For a greener future
Engineering the Plastid
Sachin S RawatSchool of Biotech, GGS IP University
A Look at the Plastid
A Plastid is a….
Major organelle of plant and algal cellsSite of manufacture and storage of
important chemical compoundsHas circular, dsDNA copiesReplicates autonomously of the cellThought to have been originated from
endosymbiotic bacteria
Plastid genes show maternal inheritance
Derived from proplastids in meristem
Have diverse functions
Chloroplasts – green plastids – for photosynthesis
Chromoplasts – coloured plastids – for pigment synthesis and storage
Gerontoplasts – control dismantling of photosynthetic apparatus during senescence
Leucoplasts – colourless plastids – monoterpene synthesis
Leucoplasts include amyloplasts (starch), elaioplasts (fats), proteinoplasts (proteins) and tannosomes (tannins)
120-130 plastid genes
Are densely packed and fall into 2 categories:
Photosynthesis-related genesGenetic system genes - genes for rRNAs,
tRNAs, ribosomal proteins and RNA polymerase subunits
A Fresher Look at the Plastid
Why plastid transformation?
High protein expression levels
Absence of epigenetic effects
Uniparental inheritance is commercially favoured
Easy transgene stacking in operons
Increased biosafety – Since plastids are maternally inherited, they aren’t transmitted by pollen
Hurdles to ‘transplastomic’ plants
Difficulty in delivering foreign DNA through double membrane of the plastid
The enormous copy number (polyploidy) of the plastid genome
The desired genetic modification must be in each copy of plastid genome in each cell
Failure to achieve homoplasmy results in rapid somatic segregation and genetic instability
Repeated rounds of selection and regeneration are required
DNA delivery into plastids2 successful methods include biolistics and
polyethylene glycol-mediated transferBiolistics is preferred as it is less time-
consuming and demanding
Integration of foreign DNA into plastid genome occurs via homologous recombination
Homologous recombination operates in plastids at a high efficiency
Biolistic chloroplast transformation and transgene integration into theplastid genome via homologous recombination
Recent success Expression of Bt toxin gene from the tobacco
plastid genomeHigh accumulation levels of Bt toxin protein (3-5 %
of TSP)Plants with high-level resistance to herbivorous
insects
Co-expression with upstream ORFs further increased Bt toxin accumulation and even resulted in its crystallization in chloroplast
Production of somatotropin (7% TSP) in tobacco plastids
Case Study I – Lactuca sativa
Protoplast isolationLettuce seeds were sterilized and sown on
MS medium with 2% sucroseShoot tips from leaves obtained were
transferred to MS medium with 3% sucroseThe leaves were cut into pieces and
incubated in PG solution, followed by enzyme solution consisting of 1% cellulase and .25% macerozyme
Protoplast suspension was filtered through nylon mesh
Protoplasts were collected at surface after centrifugation at 70g for 8min
Transformation and culture
10µl transforming DNA and 0.6ml PEG solution was added to protoplast suspension and incubated at 25ºC for 10min
Protoplasts were mixed with 1:1 solution of B5 and 2% agarose to a density of 3.6 X 104 protoplasts per ml
The suspension was plated onto Petri dishes and cultured at 25ºC in the dark
Selection was initiated on the 7th day by fresh medium containing spectinomycin dihydrochloride
AnalysesPCR – specific primers were used to assess
the presence of aadA gene in resistant cell lines
Immunoblot analysis – using HRP-conjugated secondary antibodies
Southern and Northern blots were performed to look for target genes and their transcripts
After 2 weeks, non-transformants were yellow while spectinomycin-resistant seedlings were green and growing vigorously
100% of spectinomycin-resistant lettuce cell lines were true plastid transformants
A limitation was the high frequency of polyploid cell lines
Production of humantherapeutic proteins
Why lettuce is favoured over tobacco? Most of the plant is leaf tissue and this tissue
contains the greatest number of plastids per cell
Unlike tobacco, lettuce has no toxic alkaloids that need to be removed - low purification and downstream processing costs
Lettuce is a relevant human foodstuff that can be consumed without cooking
Case Study II – Petunia hybrida
Plastid transformation
Leaf pieces were placed on MS medium supplemented with 1 mg/l 6-benzylaminopurine, 0.1 mg/l IAA, 30 g/l sucrose and 0.8% agar (MSB30)
Leaves were bombarded with 1µm, vector-coated gold particles from distance of 6cm
Incubated in dim light for 48h at 25ºCLeaves were transferred to MSB30 medium
with 200mg/l each of streptomycin sulfate and spectinomycin dihydrochloride pentahydrate
Resistant shoots first appeared after 8 weeks
Vector design
AnalysesDNA blot – gene specific primers were usedGUS assay – 5-Bromo-4-chloro-3-
indolylbeta-D-glucuronic acid was used to compare the protein expression levels between the wild type and the transformants by detecting fluorescence
Selection on two antibiotics overcomes the problem of spontaneous resistant mutants associated with using spectinomycin alone
Comparing plastid transformants with non-transformants
Good model to study plastid biology
N. tabacum is amphi-diploidA. thaliana doesn’t give rise to fertile
transplastomes
These limitations are overcome in Petunia as:
P. hybrida is diploidSuitable for mutation screening to identify
nuclear loci affecting the maintenance and expression of plastid transgenes
A Look at the Future
Metabolic pathways into plastids?
Cost-effective production platform for pharmaceuticals and nutraceuticals
Production of trehalose in tobacco chloroplasts
Tryptophan overproduction by feedback-insensitive synthesis of α-subunit of anthranilate synthase
Simplifying technology, extending crop range
Can we engineer photosynthesis?
Efficiency of photosynthesis
The most abundant protein in the world
Its CO2:O2 specificity that matters
Getting a better RubisCO for your plant
Equally precise tools for nuclear genome required
Plastids for Synthetic Biology
A compact, minimal genome
High transgene expression and low cost ideal for synthetic biology
Designing totally new plastids
References
Bock and Khan; Taming plastids for a green future; Trends in Biotechnology
Lelivelt et al.; Stable plastid transformation in lettuce; Plant Molecular Biology
Zuilen et al.; Stable transformation of Petunia plastids; Transgenic Research
Thank you