Synthetic Biology in plants

Mallikarjun P.K.
PGS20AGR8299
II Ph.D.
2
Synthetic Biology in Plant:
Towards engineered plant breeding

Flow of Seminar
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➢ Synthetic Biology (SB) – An introduction
➢ Approaches and tools for SB
➢ SB in Plants - Pioneer examples
➢ Case studies
➢ SBOL
➢ Regulatory measures
➢ Pros and Cons
➢ Conclusion

14-May-22 Department of Genetics and Plant Breeding
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van Westen and Dijkstra, 2021
A
B
Introduction

Introduction
70% increase in food production is required
Department of Genetics and Plant Breeding
An estimated report on population growth
by US census
AFW, 2016
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Timeline - How genetic manipulation has evolved

Funding for synthetic biology companies
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Department of Genetics and Plant Breeding 7

Synthetic biology funding:
Application v/s Tools and technologies
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• Gene stacking- difficult
• Transgene position effects
First Generation Genetic Engineering
Second Generation Genetic Engineering
Synthetic Biology
Need.....???
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1. First wave in synthetic biology
Genetic toggle switch, Repressilator circuits
and microbial synthetic biology
2. Second wave in synthetic biology
Plant synthetic biology
Cook et al., 2013

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Genetic engineering Synthetic biology
➢It is a process that uses
laboratory-based technologies
to alter the DNA makeup of an
organism
➢It is the design and construction
of existing biological systems
using alternative approaches.
➢An engineering-based principles
plus mathematical modeling for
designing, constructing and
testing of a completely new
genetic system.
➢new biological entities such as
enzymes, genetic circuits, and
cells or the redesign of existing
biological system.
“Genetic engineering is a tool for synthetic biology”

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The knowledge of system biology guides the design
of synthetic biology tools, which can in turn provide
insights to system biology.
Liu et al., 2013
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Plant synthetic biology is an emerging field that
aims to combines engineering principles with plant biology
towards the design and alteration of natural systems or to
the de novo construction of artificial biological devices and
systems that exhibit predictable behaviors.
Liu et al., 2015

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Dr. Eric T. Kool
(Redesign of life)

Synthesized a synthetic
bacterium genome, paving
the way for a new field of
study
Working in producing
biofuels using synthetic
biology
• He co-founded the
BioBricks Foundation
and co-organized the
iGEM
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Developed Polony
seq (NGS)
Contributed to HGP
Pioneer’s of this new technology
George Church
J. Craig Venter
Jay D. Keasling
Drew Endy

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Approaches of Synthetic biology
Wikmark et al., 2016

Design cycle for plant synthetic biology driven by
engineering principles
Lu and Stewart, 2015

Decoupling :- It allows simplification of complex problems into
many smaller problems that can be addressed individually.
Abstraction:- It separates topologies of information into
hierarchical levels (such as DNA, parts, devices, and
systems.) and allows limited and principled information
exchanges between levels.
Standardization :- It is used to define and characterize
orthogonal parts and standardized conditions for testing in a
complex system.
Engineering principles for design
Drew et al., 2005

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Abstraction hierarchy allows the breakdown of complexity
Andrianantoandro et al., 2006

Biobricks – parts used in synthetic biology
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➢ It is a trademark term for man-made DNA sequences
encoding elementary modules that may be combined to
produce more complex synthetic biological systems.
➢Tom Knight at MIT’s CSAIL in 2003.

Components for plant synthetic devices
➢ In biological systems parts include cis-regulatory elements,
promoters, transcription IS, exons, protein domains, ORFs and
terminators can be reduced into parts and modules for
reconstruction.
➢ Synthetic parts can be assembled modularly resulting in
different hierarchical functions, such as synthetic genes,
pathways, chromosomes, genomes, conglomerate biological
devices and networks.
➢ These synthetic devices can operate at various levels in the
Central Dogma and beyond, such as at transcriptional,
translational, and post-translational levels.

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Genes are like Components Parts in a
circuit board
Promoter Terminator
Coding Region
New biological “devices” can be built by combing parts.
Several units that comprise entire metabolic pathway are
assembled and introduced into an organism.
“Transcriptors”
Xin et al., 2019

Tools for design and modeling
Component design and synthesis
➢ Gene Design, Gene Designer 2.0
Topology and network design
➢ GenoCAD, SynBioSS, CellDesigner
Simulation and Behavior prediction
➢ CellModeller, COPASI
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CAD software for syntheticbiology

DBTL engineering cycle used in synthetic biology
Meng and Ellis,
2020

Pioneer examples of
synthetic biology

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Switch-OFF kinetics of red light-regulated
gene expression in tobacco

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Synthetic genome annotation map
1.08 Mb
Mycoplasmamycoides
JCVI-syn1.0 genome

Characterization of the synthetic genome
WM – DNA Water Mark

Case Study - I

The BHAC as photorespiratory bypass in plant
peroxisomal matrix

Material and methods
➢ A. thaliana - wild type (WT) Col-0 and mutant type - ggt1-1
➢ BHAC genes were codon optimized for expression in A.
thaliana
➢ In total, 14 and 11 primary transformants were obtained
Out of which four BHAC lines, (Col::BHAC #1 and #2) and
(ggt1-1::BHAC #1 and #2) respectively are used in this study
➢ BHAC Enzyme Activity : Immuno blot assay
➢ Metabolic profiling : GC/MS Q-TOF and IC/MS method.
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BHAC enzymes activity in Arabidopsis
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Results

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➢The formation of BHAC-specific metabolites shows that the
peroxisomal BHAC indeed functions as photorespiratory bypass.
Relative metabolite levels in BHAC plants grown in
ambient air

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Metabolome profiles of ambient air grown plants
BHAC reshapes the
metabolome in plants
by altering nitrogen
metabolism

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Representative images of BHAC plants showing reduces plant growth
Relative levels of phosphorylated metabolites in BHAC plants
grown in ambient air.

Conclusion
➢ The function of synthetically engineered BHAC in Arabidopsis
peroxisomes, demonstrated that a photorespiratory bypasses
can improve plant yield
➢ BHAC in the ggt1-1 mutant pushes pathway and improved
plant growth compared to the mutant background.
➢ BHAC functions as a nitrogen-conserving pathway and allows
rerouting of photorespiratory glycolate into amino acids.
➢ By engineering two of the main targets in primary plant
metabolism, this study creates opportunities for improved
agricultural productivity in the future.

Case Study - II
Proceedings of the NationalAcademy of Sciences, 117(28), pp.16537-45.

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➢ Stable expression of each component of the nitrogenase system
in an active form is a prerequisite for engineering nitrogen
fixation in eukaryotic cells.
➢ Mitochondria provide an oxygen depleted environment for the
expression of active nitrogenase in plants.
➢ NifD, is susceptible to cleavage by mitochondrial processing
peptidases, presenting a major challenge to engineering nitrogen
fixation in mitochondria.
➢ The development of synthetic biology tools has enabled a
potential solution for engineering active MoFe protein in plants.
Introduction

Strains used
E. coli stains :- Top10, JM109 and NCM3722
S. cerevisiae :- W303-1a
Media used
LB broth - E. coli growth
KPM minimal medium - Diazotropic growth
YDS medium - S. cerevisiae growth
Synthetic dropout medium - used to select colony after
transformation
YPDG medium - used for protein expression studies in S.
cerevisiae
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Material and methods
Klebsiella oxytoca

Methods used
➢ Plasmid construction - using golden gate assembly
➢ Acetylene Reduction assay and Diazotrophic growth.
➢ Stability assay of the Tag-labeled Nif components.
➢ Western blot assays.

Results
Identification of a key amino acid residue for NifD degradation

Screening for stable NifD R98 variants that retain Nitrogenase
functionality

The crystal structure of K.
oxytoca nitrogenase MoFe protein
showing interactions between
NifD R98 with adjecent residues.
(PDB ID code 1QGU)

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Determination of the stability of Nif proteins
Intact NifD
Intact NifD
Intact NifD
Processed
NifDn
Processed
NifDn
Processed
NifDn

➢ They predicted that R98 region which is conserved among diverse
NifD proteins is responsible for protease mediated cleavage.
➢ They confirms unequivocally that the MPP was responsible for the
cleavage of NifD proteins.
➢ The R98P mutation of NifD protein could enable universal
application in engineering of stable nitrogenase in plant
mitochondria.
➢ By this approach non-legume crops can “fix” their own nitrogen so
that it reduces the use of industrial nitrogen fertilizers in
agriculture.
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Conclusion

Synthetic Biology Open Language (SBOL)
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➢ It is a free and open source standard for insilico
representation of structural and basic qualitative behavioral
aspects of a biological designs.
➢ It was developed by the synthetic biology community to
create a standardized format of biological designs.

Who is developing synthetic biology ???
Wang et al., 2021
Student teams design, build and test their projects and gather to present
their work and compete at the annual gatherings.
It is a growing biotechnological social movement, which allows numbers
of small organizations and individuals to participate in R & D, with
spreading knowledge a higher priority than turning profits.

Regulatory measures
➢ Intellectual property rights for synthetic biology can be
described as a potential “perfect storm”
➢ an Ad Hoc Technical Expert Group (AHTEG) will evaluate,
regulatory framework for addressing potential impacts
from synthetic biology technologies.
➢ The iGEM’s Registry of Standard Biological Parts,
contributing researchers post their BioBrick™ parts on
public domain
➢ Currently DBT saying to Centre that the need of National
policy on synthetic biology, an emerging science.

Applications
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Future products of synthetic biology
Anti-malaria drug
Glowing plant
Vanilla made from
yeast
Oils from modified
algae

14-May-22 Roell and Zurbriggen, 2020
Impact of SB on future
Agriculture and
Nutrition

Limitations
➢Plant synthetic biology is currently slow process,
costly, and technically challenged
➢Biological systems are infinitely more complex and do
not behave in a linearly predictable way
➢Bioterrorism
➢Recreation of extincted pathogens
➢Ethical concerns due to further evolution of DNA’s
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➢ R & D needed on DBTL for accelerated plant synthetic biology.
➢ Developing larger libraries, algorithms, models and software's
of biological parts and modules.
➢ Support to plant synthetic biology projects have to be started by
funding agencies in various countries.
➢ synthetic biology requires greater biosafety, biosecurity and
cyber-biosecurity.
➢ A frequent communication needed among scientists, regulators,
sociologists and other interested parties in applications of
synthetic biology.
Overcomes of limitations

Questions need to be answer
➢ How can we scale up the current simple devices to larger and
more complex synthetic biological systems
➢ How can computational models for design and simulation be
significantly improved so that synthetic circuits can better meet
inherent biological variability, uncertainty and evolution
➢ How will the products of plant synthetic biology be regulated
➢ Can the plant science community and bioethicists effectively
communicate the risks and benefits of new applications and
products to consumers
14-May-22

Thank you
55
14-May-22

Synthetic Biology in plants

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