Computer-aided design (CAD) is the use of computers (or workstations) to aid in the creation, modification, analysis, or optimization of a design.
CAD software is used to increase the productivity of the designer,
improve the quality of design, improve communications through
documentation, and to create a database for manufacturing. Designs made through CAD software are helpful in protecting products and inventions when used in patent
applications. CAD output is often in the form of electronic files for
print, machining, or other manufacturing operations. The term CADD (for computer aided design and drafting) is also used.
CAD software for mechanical design uses either vector-based
graphics to depict the objects of traditional drafting, or may also
produce raster graphics showing the overall appearance of designed objects. However, it involves more than just shapes. As in the manual drafting of technical and engineering drawings, the output of CAD must convey information, such as materials, processes, dimensions, and tolerances, according to application-specific conventions.
CAD may be used to design curves and figures in two-dimensional (2D) space; or curves, surfaces, and solids in three-dimensional (3D) space.
The design of geometric models for object shapes, in particular, is occasionally called computer-aided geometric design (CAGD).
Overview of CAD software
Starting
around the mid-1960s, with the IBM Drafting System, computer-aided
design systems began to provide more capability than just an ability to
reproduce manual drafting with electronic drafting, the cost-benefit for
companies to switch to CAD became apparent. The benefits of CAD
systems over manual drafting are the capabilities one often takes for
granted from computer systems today; automated generation of bills of materials, auto layout in integrated circuits,
interference checking, and many others. Eventually, CAD provided the
designer with the ability to perform engineering calculations. During
this transition, calculations were still performed either by hand or by
those individuals who could run computer programs. CAD was a
revolutionary change in the engineering industry, where draftsmen,
designers, and engineering roles begin to merge. It did not eliminate
departments as much as it merged departments and empowered draftsmen,
designers, and engineers. CAD is an example of the pervasive effect
computers were beginning to have on the industry.
Current computer-aided design software packages range from 2D vector-based drafting systems to 3D solid and surface
modelers. Modern CAD packages can also frequently allow rotations in
three dimensions, allowing viewing of a designed object from any desired
angle, even from the inside looking out. Some CAD software is capable
of dynamic mathematical modeling.
CAD technology is used in the design of tools and machinery and
in the drafting and design of all types of buildings, from small
residential types (houses) to the largest commercial and industrial
structures (hospitals and factories).
CAD is mainly used for detailed engineering of 3D models or 2D
drawings of physical components, but it is also used throughout the
engineering process from conceptual design and layout of products,
through strength and dynamic analysis of assemblies to definition of
manufacturing methods of components. It can also be used to design
objects such as jewelry, furniture, appliances, etc. Furthermore, many
CAD applications now offer advanced rendering and animation capabilities
so engineers can better visualize their product designs. 4D BIM
is a type of virtual construction engineering simulation incorporating
time or schedule-related information for project management.
CAD has become an especially important technology within the scope of computer-aided technologies, with benefits such as lower product development costs and a greatly shortened design cycle.
CAD enables designers to layout and develop work on screen, print it
out and save it for future editing, saving time on their drawings.
Uses
Computer-aided design is one of the many tools used by engineers and
designers and is used in many ways depending on the profession of the
user and the type of software in question.
CAD is one part of the whole digital product development (DPD) activity within the product lifecycle management
(PLM) processes, and as such is used together with other tools, which
are either integrated modules or stand-alone products, such as:
CAD is also used for the accurate creation of photo simulations that
are often required in the preparation of environmental impact reports,
in which computer-aided designs of intended buildings are superimposed
into photographs of existing environments to represent what that locale
will be like, where the proposed facilities are allowed to be built.
Potential blockage of view corridors and shadow studies are also
frequently analyzed through the use of CAD.
CAD has been proven to be useful to engineers as well. Using four properties which are history, features, parameterization,
and high-level constraints. The construction history can be used to
look back into the model's personal features and work on the single area
rather than the whole model. Parameters and constraints can be used to
determine the size, shape, and other properties of the different
modeling elements. The features in the CAD system can be used for the
variety of tools for measurement such as tensile strength, yield
strength, electrical, or electromagnetic properties. Also its stress,
strain, timing, or how the element gets affected in certain temperatures, etc.
Types
A simple procedure of recreating a solid model out of 2D sketches.
There are several different types of CAD,
each requiring the operator to think differently about how to use them
and design their virtual components in a different manner for each.
There are many producers of the lower-end 2D systems, including a
number of free and open-source programs. These provide an approach to
the drawing process without all the fuss over scale and placement on the
drawing sheet that accompanied hand drafting since these can be
adjusted as required during the creation of the final draft.
3D wireframe
is basically an extension of 2D drafting (not often used today). Each
line has to be manually inserted into the drawing. The final product has
no mass properties associated with it and cannot have features directly
added to it, such as holes. The operator approaches these in a similar
fashion to the 2D systems, although many 3D systems allow using the
wireframe model to make the final engineering drawing views.
3D "dumb" solids are created in a way analogous to
manipulations of real-world objects (not often used today). Basic
three-dimensional geometric forms (prisms, cylinders, spheres, and so
on) have solid volumes added or subtracted from them as if assembling or
cutting real-world objects. Two-dimensional projected views can easily
be generated from the models. Basic 3D solids don't usually include
tools to easily allow motion of components, set limits to their motion,
or identify interference between components.
Parametric modeling allows the operator to use what is
referred to as "design intent". The objects and features created are
modifiable. Any future modifications can be made by changing how the
original part was created. If a feature was intended to be located from
the center of the part, the operator should locate it from the center of
the model. The feature could be located using any geometric object
already available in the part, but this random placement would defeat
the design intent. If the operator designs the part as it functions the
parametric modeler is able to make changes to the part while maintaining
geometric and functional relationships.
Direct or explicit modeling
provide the ability to edit geometry without a history tree. With
direct modeling, once a sketch is used to create geometry the sketch is
incorporated into the new geometry and the designer just modifies the
geometry without needing the original sketch. As with parametric
modeling, direct modeling has the ability to include relationships between selected geometry (e.g., tangency, concentricity).
Top-end systems offer the capabilities to incorporate more organic, aesthetic, and ergonomic features into designs. Freeform surface modeling
is often combined with solids to allow the designer to create products
that fit the human form and visual requirements as well as they
interface with the machine.
Originally software for CAD systems was developed with computer languages such as Fortran, ALGOL but with the advancement of object-oriented programming methods this has radically changed. Typical modern parametric feature-based modeler and freeform surface systems are built around a number of key C modules with their own APIs. A CAD system can be seen as built up from the interaction of a graphical user interface (GUI) with NURBS geometry or boundary representation (B-rep) data via a geometric modeling kernel.
A geometry constraint engine may also be employed to manage the
associative relationships between geometry, such as wireframe geometry
in a sketch or components in an assembly.
Unexpected capabilities of these associative relationships have led to a new form of prototyping called digital prototyping.
In contrast to physical prototypes, which entail manufacturing time in
the design. That said, CAD models can be generated by a computer after
the physical prototype has been scanned using an industrial CT scanning
machine. Depending on the nature of the business, digital or physical
prototypes can be initially chosen according to specific needs.
Today, CAD systems exist for all the major platforms (Windows, Linux, UNIX and Mac OS X); some packages support multiple platforms.
Currently, no special hardware is required for most CAD software.
However, some CAD systems can do graphically and computationally
intensive tasks, so a modern graphics card, high speed (and possibly multiple) CPUs and large amounts of RAM may be recommended.
The human-machine interface is generally via a computer mouse but can also be via a pen and digitizing graphics tablet. Manipulation of the view of the model on the screen is also sometimes done with the use of a Spacemouse/SpaceBall. Some systems also support stereoscopic glasses for viewing the 3D model.
Technologies which in the past were limited to larger installations or
specialist applications have become available to a wide group of users.
These include the CAVE or HMDs and interactive devices like motion-sensing technology
Software
CAD software enables engineers and architects to design, inspect and manage engineering projects within an integrated graphical user interface (GUI) on a personal computer system. Most applications support solid modeling with boundary representation (B-Rep) and NURBS geometry, and enable the same to be published in a variety of formats. A geometric modeling kernel is a software component that provides solid modeling and surface modeling features to CAD applications.
Based on market statistics, commercial software from Autodesk, Dassault Systems, Siemens PLM Software, and PTC dominate the CAD industry. The following is a list of major CAD applications, grouped by usage statistics.
Synthetic biology (SynBio) is a multidisciplinary area
of research that seeks to create new biological parts, devices, and
systems, or to redesign systems that are already found in nature.
Due to more powerful genetic engineering capabilities and decreased DNA synthesis and sequencing costs,
the field of synthetic biology is rapidly growing. In 2016, more than
350 companies across 40 countries were actively engaged in synthetic
biology applications; all these companies had an estimated net worth of
$3.9 billion in the global market.
Definition
Synthetic biology currently has no generally accepted definition. Here are a few examples:
"the use of a mixture of physical engineering and genetic engineering to create new (and, therefore, synthetic) life forms"
"an emerging field of research that aims to combine the knowledge
and methods of biology, engineering and related disciplines in the
design of chemically synthesized DNA to create organisms with novel or
enhanced characteristics and traits"
“applying the engineering paradigm of systems design to biological
systems in order to produce predictable and robust systems with novel
functionalities that do not exist in nature” (The European Commission,
2005) This can include the possibility of a molecular assembler, based upon biomolecular systems such as the ribosome
Synthetic biology has traditionally been divided into two different approaches: top down and bottom up.
The top down approach involves using metabolic and genetic engineering techniques to impart new functions to living cells.
The bottom up approach involves creating new biological systems in vitro by bringing together 'non-living' biomolecular components, often with the aim of constructing an artificial cell.
Biological systems are thus assembled module-by-module. Cell-free protein expression systems are often employed,
as are membrane-based molecular machinery. There are increasing
efforts to bridge the divide between these approaches by forming hybrid
living/synthetic cells, and engineering communication between living and synthetic cell populations.
History
1910: First identifiable use of the term "synthetic biology" in Stéphane Leduc's publication Théorie physico-chimique de la vie et générations spontanées. He also noted this term in another publication, La Biologie Synthétique in 1912.
1961: Jacob and Monod postulate cellular regulation by molecular networks from their study of the lac operon in E. coli and envisioned the ability to assemble new systems from molecular components.
1973: First molecular cloning and amplification of DNA in a plasmid is published in P.N.A.S. by Cohen, Boyer et al. constituting the dawn of synthetic biology.
The work on restriction nucleases not only permits us
easily to construct recombinant DNA molecules and to analyze individual
genes, but also has led us into the new era of synthetic biology where
not only existing genes are described and analyzed but also new gene
arrangements can be constructed and evaluated.
1988: First DNA amplification by the polymerase chain reaction (PCR) using a thermostable DNA polymerase is published in Science by Mullis et al. This obviated adding new DNA polymerase after each PCR cycle, thus greatly simplifying DNA mutagenesis and assembly.
2003: The most widely used standardized DNA parts, BioBrick plasmids, are invented by Tom Knight.
These parts will become central to the international Genetically
Engineered Machine competition (iGEM) founded at MIT in the following
year.
2003: Researchers engineer an artemisinin precursor pathway in E. coli.
2004: First international conference for synthetic
biology, Synthetic Biology 1.0 (SB1.0) is held at the Massachusetts
Institute of Technology, USA.
2005: Researchers develop a light-sensing circuit in E. coli. Another group designs circuits capable of multicellular pattern formation.
2006: Researchers engineer a synthetic circuit that promotes bacterial invasion of tumour cells.
2010: Researchers publish in Science the first synthetic bacterial genome, called M. mycoides JCVI-syn1.0. The genome is made from chemically-synthesized DNA using yeast recombination.
2011: Functional synthetic chromosome arms are engineered in yeast.
2012: Charpentier and Doudna labs publish in Science the programming of CRISPR-Cas9 bacterial immunity for targeting DNA cleavage. This technology greatly simplified and expanded eukaryotic gene editing.
Engineers view biology as a technology (in other words, a given system includes biotechnology or its biological engineering)
Synthetic biology includes the broad redefinition and expansion of
biotechnology, with the ultimate goals of being able to design and build
engineered live biological systems that process information, manipulate
chemicals, fabricate materials and structures, produce energy, provide
food, and maintain and enhance human health, as well as advance
fundamental knowledge of biological systems and our environment.
Studies in synthetic biology can be subdivided into broad
classifications according to the approach they take to the problem at
hand: standardization of biological parts, biomolecular engineering,
genome engineering, metabolic engineering.
Biomolecular engineering includes approaches that aim to create a
toolkit of functional units that can be introduced to present new
technological functions in living cells. Genetic engineering includes approaches to construct synthetic chromosomes or minimal organisms like Mycoplasma laboratorium.
Biomolecular design refers to the general idea of de novo design
and additive combination of biomolecular components. Each of these
approaches share a similar task: to develop a more synthetic entity at a
higher level of complexity by inventively manipulating a simpler part
at the preceding level.
On the other hand, "re-writers" are synthetic biologists
interested in testing the irreducibility of biological systems. Due to
the complexity of natural biological systems, it would be simpler to
rebuild the natural systems of interest from the ground up; In order to
provide engineered surrogates that are easier to comprehend, control and
manipulate. Re-writers draw inspiration from refactoring, a process sometimes used to improve computer software.
Enabling technologies
Several novel enabling technologies were critical to the success of synthetic biology. Concepts include standardization of biological parts and hierarchical abstraction to permit using those parts in synthetic systems.
Basic technologies include reading and writing DNA (sequencing and
fabrication). Measurements under multiple conditions are needed for
accurate modeling and computer-aided design (CAD).
DNA and gene synthesis
Driven by dramatic decreases in costs of oligonucleotide ("oligos") synthesis and the advent of PCR, the sizes of DNA constructions from oligos have increased to the genomic level. In 2000, researchers reported synthesis of the 9.6 kbp (kilo bp) Hepatitis C virus genome from chemically synthesized 60 to 80-mers. In 2002 researchers at Stony Brook University succeeded in synthesizing the 7741 bp poliovirus genome from its published sequence, producing the second synthetic genome, spanning two years. In 2003 the 5386 bp genome of the bacteriophagePhi X 174 was assembled in about two weeks. In 2006, the same team, at the J. Craig Venter Institute, constructed and patented a synthetic genome of a novel minimal bacterium, Mycoplasma laboratorium and were working on getting it functioning in a living cell.
In 2007 it was reported that several companies were offering synthesis of genetic sequences up to 2000 base pairs (bp) long, for a price of about $1 per bp and a turnaround time of less than two weeks. Oligonucleotides harvested from a photolithographic- or inkjet-manufactured DNA chip combined with PCR and DNA mismatch error-correction allows inexpensive large-scale changes of codons in genetic systems to improve gene expression or incorporate novel amino-acids (see George M. Church's and Anthony Forster's synthetic cell projects.) This favors a synthesis-from-scratch approach.
Additionally, the CRISPR/Cas
system has emerged as a promising technique for gene editing. It was
described as "the most important innovation in the synthetic biology
space in nearly 30 years". While other methods take months or years to edit gene sequences, CRISPR speeds that time up to weeks. Due to its ease of use and accessibility, however, it has raised ethical concerns, especially surrounding its use in biohacking.
Sequencing
DNA sequencing determines the order of nucleotide
bases in a DNA molecule. Synthetic biologists use DNA sequencing in
their work in several ways. First, large-scale genome sequencing efforts
continue to provide information on naturally occurring organisms. This
information provides a rich substrate from which synthetic biologists
can construct parts and devices. Second, sequencing can verify that the
fabricated system is as intended. Third, fast, cheap, and reliable
sequencing can facilitate rapid detection and identification of
synthetic systems and organisms.
Microfluidics
Microfluidics,
in particular droplet microfluidics, is an emerging tool used to
construct new components, and to analyse and characterize them. It is widely employed in screening assays.
While DNA is most important for information storage, a large
fraction of the cell's activities are carried out by proteins. Tools can
send proteins to specific regions of the cell and to link different
proteins together. The interaction strength between protein partners
should be tunable between a lifetime of seconds (desirable for dynamic
signaling events) up to an irreversible interaction (desirable for
device stability or resilient to harsh conditions). Interactions such as
coiled coils, SH3 domain-peptide binding or SpyTag/SpyCatcher offer such control. In addition it is necessary to regulate protein-protein interactions in cells, such as with light (using light-oxygen-voltage-sensing domains) or cell-permeable small molecules by chemically induced dimerization.
In a living cell, molecular motifs are embedded in a bigger
network with upstream and downstream components. These components may
alter the signaling capability of the modeling module. In the case of
ultrasensitive modules, the sensitivity contribution of a module can
differ from the sensitivity that the module sustains in isolation.
Modeling
Models
inform the design of engineered biological systems by better predicting
system behavior prior to fabrication. Synthetic biology benefits from
better models of how biological molecules bind substrates and catalyze
reactions, how DNA encodes the information needed to specify the cell
and how multi-component integrated systems behave. Multiscale models of
gene regulatory networks focus on synthetic biology applications.
Simulations can model all biomolecular interactions in transcription, translation, regulation and induction of gene regulatory networks.
Synthetic transcription factors
Studies have considered the components of the DNA transcription mechanism. One desire of scientists creating synthetic biological circuits is to be able to control the transcription of synthetic DNA in unicellular organisms (prokaryotes) and in multicellular organisms (eukaryotes). One study tested the adjustability of synthetic transcription factors (sTFs) in areas of transcription output and cooperative ability among multiple transcription factor complexes. Researchers were able to mutate functional regions called zinc fingers,
the DNA specific component of sTFs, to decrease their affinity for
specific operator DNA sequence sites, and thus decrease the associated
site-specific activity of the sTF (usually transcriptional regulation).
They further used the zinc fingers as components of complex-forming
sTFs, which are the eukaryotic translation mechanisms.
Applications
Biological computers
A biological computer
refers to an engineered biological system that can perform
computer-like operations, which is a dominant paradigm in synthetic
biology. Researchers built and characterized a variety of logic gates in a number of organisms,
and demonstrated both analog and digital computation in living cells.
They demonstrated that bacteria can be engineered to perform both analog
and/or digital computation. In human cells research demonstrated a universal logic evaluator that operates in mammalian cells in 2007.
Subsequently, researchers utilized this paradigm to demonstrate a
proof-of-concept therapy that uses biological digital computation to
detect and kill human cancer cells in 2011. Another group of researchers demonstrated in 2016 that principles of computer engineering, can be used to automate digital circuit design in bacterial cells.
In 2017, researchers demonstrated the 'Boolean logic and arithmetic
through DNA excision' (BLADE) system to engineer digital computation in
human cells.
Biosensors
A biosensor
refers to an engineered organism, usually a bacterium, that is capable
of reporting some ambient phenomenon such as the presence of heavy
metals or toxins. One such system is the Lux operon of Aliivibrio fischeri, which codes for the enzyme that is the source of bacterial bioluminescence, and can be placed after a respondent promoter to express the luminescence genes in response to a specific environmental stimulus. One such sensor created, consisted of a bioluminescent bacterial coating on a photosensitive computer chip to detect certain petroleumpollutants. When the bacteria sense the pollutant, they luminesce. Another example of a similar mechanism is the detection of landmines by an engineered E.coli reporter strain capable of detecting TNT and its main degradation product DNT, and consequently producing a green fluorescent protein (GFP).
Modified organisms can sense environmental signals and send
output signals that can be detected and serve diagnostic purposes.
Microbe cohorts have been used.
Cell transformation
Cells
use interacting genes and proteins, which are called gene circuits, to
implement diverse function, such as responding to environmental signals,
decision making and communication. Three key components are involved:
DNA, RNA and Synthetic biologist designed gene circuits that can control
gene expression from several levels including transcriptional,
post-transcriptional and translational levels.
Traditional metabolic engineering has been bolstered by the
introduction of combinations of foreign genes and optimization by
directed evolution. This includes engineering E. coli and yeast for commercial production of a precursor of the antimalarial drug, Artemisinin.
Entire organisms have yet to be created from scratch, although living cells can be transformed with new DNA. Several ways allow constructing synthetic DNA components and even entire synthetic genomes,
but once the desired genetic code is obtained, it is integrated into a
living cell that is expected to manifest the desired new capabilities or
phenotypes while growing and thriving. Cell transformation is used to create biological circuits, which can be manipulated to yield desired outputs.
By integrating synthetic biology with materials science,
it would be possible to use cells as microscopic molecular foundries to
produce materials with properties whose properties were genetically
encoded. Re-engineering has produced Curli fibers, the amyloid component of extracellular material of biofilms, as a platform for programmable nanomaterial.
These nanofibers were genetically constructed for specific functions,
including adhesion to substrates, nanoparticle templating and protein
immobilization.
Designed proteins
The Top7 protein was one of the first proteins designed for a fold that had never been seen before in nature
Natural proteins can be engineered, for example, by directed evolution,
novel protein structures that match or improve on the functionality of
existing proteins can be produced. One group generated a helix bundle that was capable of binding oxygen with similar properties as hemoglobin, yet did not bind carbon monoxide. A similar protein structure was generated to support a variety of oxidoreductase activities while another formed a structurally and sequentially novel ATPase. Another group generated a family of G-protein coupled receptors that could be activated by the inert small molecule clozapine N-oxide but insensitive to the native ligand, acetylcholine; these receptors are known as DREADDs.
Novel functionalities or protein specificity can also be engineered
using computational approaches. One study was able to use two different
computational methods – a bioinformatics and molecular modeling method
to mine sequence databases, and a computational enzyme design method to
reprogram enzyme specificity. Both methods resulted in designed enzymes
with greater than 100 fold specificity for production of longer chain
alcohols from sugar.
Another common investigation is expansion of the natural set of 20 amino acids. Excluding stop codons, 61 codons
have been identified, but only 20 amino acids are coded generally in
all organisms. Certain codons are engineered to code for alternative
amino acids including: nonstandard amino acids such as O-methyl tyrosine; or exogenous amino acids such as 4-fluorophenylalanine. Typically, these projects make use of re-coded nonsense suppressortRNA-Aminoacyl tRNA synthetase pairs from other organisms, though in most cases substantial engineering is required.
Other researchers investigated protein structure and function by
reducing the normal set of 20 amino acids. Limited protein sequence
libraries are made by generating proteins where groups of amino acids
may be replaced by a single amino acid. For instance, several non-polar amino acids within a protein can all be replaced with a single non-polar amino acid. One project demonstrated that an engineered version of Chorismate mutase still had catalytic activity when only 9 amino acids were used.
Researchers and companies practice synthetic biology to synthesize industrial enzymes
with high activity, optimal yields and effectiveness. These synthesized
enzymes aim to improve products such as detergents and lactose-free
dairy products, as well as make them more cost effective.
The improvements of metabolic engineering by synthetic biology is an
example of a biotechnological technique utilized in industry to discover
pharmaceuticals and fermentive chemicals. Synthetic biology may
investigate modular pathway systems in biochemical production and
increase yields of metabolic production. Artificial enzymatic activity
and subsequent effects on metabolic reaction rates and yields may
develop "efficient new strategies for improving cellular properties ...
for industrially important biochemical production".
Designed nucleic acid systems
Scientists can encode digital information onto a single strand of synthetic DNA. In 2012, George M. Church encoded one of his books about synthetic biology in DNA. The 5.3 Mb of data was more than 1000 times greater than the previous largest amount of information to be stored in synthesized DNA. A similar project encoded the complete sonnets of William Shakespeare in DNA. More generally, algorithms such as NUPACK, ViennaRNA, Ribosome Binding Site Calculator, Cello, and Non-Repetitive Parts Calculator enables the design of new genetic systems.
Many technologies have been developed for incorporating unnatural nucleotides and amino acids into nucleic acids and proteins, both in vitro and in vivo. For example, in May 2014, researchers announced that they had successfully introduced two new artificial nucleotides
into bacterial DNA. By including individual artificial nucleotides in
the culture media, they were able to exchange the bacteria 24 times;
they did not generate mRNA or proteins able to use the artificial nucleotides.
Space exploration
Synthetic biology raised NASA's interest as it could help to produce resources for astronauts from a restricted portfolio of compounds sent from Earth.
On Mars, in particular, synthetic biology could lead to production
processes based on local resources, making it a powerful tool in the
development of manned outposts with less dependence on Earth.
Work has gone into developing plant strains that are able to cope with
the harsh Martian environment, using similar techniques to those
employed to increase resilience to certain environmental factors in
agricultural crops.
Synthetic life
Gene functions in the minimal genome of the synthetic organism, Syn 3.
One important topic in synthetic biology is synthetic life, that is concerned with hypothetical organisms created in vitro from biomolecules and/or chemical analogues thereof. Synthetic life experiments attempt to either probe the origins of life, study some of the properties of life, or more ambitiously to recreate life from non-living (abiotic)
components. Synthetic life biology attempts to create living organisms
capable of carrying out important functions, from manufacturing
pharmaceuticals to detoxifying polluted land and water.
In medicine, it offers prospects of using designer biological parts as a
starting point for new classes of therapies and diagnostic tools.
A living "artificial cell" has been defined as a completely synthetic cell that can capture energy, maintain ion gradients, contain macromolecules as well as store information and have the ability to mutate. Nobody has been able to create such a cell.
A completely synthetic bacterial chromosome was produced in 2010 by Craig Venter, and his team introduced it to genomically emptied bacterial host cells. The host cells were able to grow and replicate. The Mycoplasma laboratorium is the only living organism with completely engineered genome.
The first living organism with 'artificial' expanded DNA code was presented in 2014; the team used E. coli that had its genome extracted and replaced with a chromosome with an expanded genetic code. The nucleosides added are d5SICS and dNaM.
In 2017 the international Build-a-Cell large-scale research collaboration for the construction of synthetic living cell was started, followed by national synthetic cell organizations in several countries, including FabriCell, MaxSynBio and BaSyC. The European synthetic cell efforts were unified in 2019 as SynCellEU initiative.
Drug delivery platforms
Engineered bacteria-based platform
Bacteria have long been used in cancer treatment. Bifidobacterium and Clostridium selectively colonize tumors and reduce their size.
Recently synthetic biologists reprogrammed bacteria to sense and
respond to a particular cancer state. Most often bacteria are used to
deliver a therapeutic molecule directly to the tumor to minimize
off-target effects. To target the tumor cells, peptides that can specifically recognize a tumor were expressed on the surfaces of bacteria. Peptides used include an affibody molecule that specifically targets human epidermal growth factor receptor 2 and a synthetic adhesin. The other way is to allow bacteria to sense the tumor microenvironment, for example hypoxia, by building an AND logic gate into bacteria. The bacteria then only release target therapeutic molecules to the tumor through either lysis or the bacterial secretion system.
Lysis has the advantage that it can stimulate the immune system and
control growth. Multiple types of secretion systems can be used and
other strategies as well. The system is inducible by external signals.
Inducers include chemicals, electromagnetic or light waves.
Multiple species and strains are applied in these therapeutics. Most commonly used bacteria are Salmonella typhimurium, Escherichia Coli, Bifidobacteria, Streptococcus, Lactobacillus, Listeria and Bacillus subtilis.
Each of these species have their own property and are unique to cancer
therapy in terms of tissue colonization, interaction with immune system
and ease of application.
Cell-based platform
The immune system plays an important role in cancer and can be harnessed to attack cancer cells. Cell-based therapies focus on immunotherapies, mostly by engineering T cells.
T cell receptors were engineered and ‘trained’ to detect cancer epitopes. Chimeric antigen receptors (CARs) are composed of a fragment of an antibody
fused to intracellular T cell signaling domains that can activate and
trigger proliferation of the cell. A second generation CAR-based therapy
was approved by FDA.
Gene switches were designed to enhance safety of the treatment.
Kill switches were developed to terminate the therapy should the patient
show severe side effects. Mechanisms can more finely control the system and stop and reactivate it.
Since the number of T-cells are important for therapy persistence and
severity, growth of T-cells is also controlled to dial the effectiveness
and safety of therapeutics.
Although several mechanisms can improve safety and control,
limitations include the difficulty of inducing large DNA circuits into
the cells and risks associated with introducing foreign components,
especially proteins, into cells.
Ethics
The creation of new life and the tampering of existing life has raised ethical concerns in the field of synthetic biology and are actively being discussed.
Common ethical questions include:
Is it morally right to tamper with nature?
Is one playing God when creating new life?
What happens if a synthetic organism accidentally escapes?
What if an individual misuses synthetic biology and creates a harmful entity (e.g., a biological weapon)?
Who will have control of and access to the products of synthetic biology?
Who will gain from these innovations? Investors? Medical patients? Industrial farmers?
Does the patent system allow patents on living organisms? What about parts of organisms, like HIV resistance genes in humans?
What if a new creation is deserving of moral or legal status?
The ethical aspects of synthetic biology has 3 main features: biosafety, biosecurity, and the creation of new life forms.
Other ethical issues mentioned include the regulation of new creations,
patent management of new creations, benefit distribution, and research
integrity.
Ethical issues have surfaced for recombinant DNA and genetically modified organism (GMO) technologies and extensive regulations of genetic engineering and pathogen research were in place in many jurisdictions. Amy Gutmann,
former head of the Presidential Bioethics Commission, argued that we
should avoid the temptation to over-regulate synthetic biology in
general, and genetic engineering in particular. According to Gutmann,
"Regulatory parsimony is especially important in emerging
technologies...where the temptation to stifle innovation on the basis of
uncertainty and fear of the unknown is particularly great. The blunt
instruments of statutory and regulatory restraint may not only inhibit
the distribution of new benefits, but can be counterproductive to
security and safety by preventing researchers from developing effective
safeguards.".
The "creation" of life
One
ethical question is whether or not it is acceptable to create new life
forms, sometimes known as "playing God". Currently, the creation of new
life forms not present in nature is at small-scale, the potential
benefits and dangers remain unknown, and careful consideration and
oversight are ensured for most studies.
Many advocates express the great potential value—to agriculture,
medicine, and academic knowledge, among other fields—of creating
artificial life forms. Creation of new entities could expand scientific
knowledge well beyond what is currently known from studying natural
phenomena. Yet there is concern that artificial life forms may reduce
nature's "purity" (i.e., nature could be somehow corrupted by human
intervention and manipulation) and potentially influence the adoption of
more engineering-like principles instead of biodiversity- and
nature-focused ideals. Some are also concerned that if an artificial
life form were to be released into nature, it could hamper biodiversity
by beating out natural species for resources (similar to how algal blooms kill marine species). Another concern involves the ethical treatment of newly created entities if they happen to sense pain, sentience, and self-perception. Should such life be given moral or legal rights? If so, how?
Biosafety and biocontainment
What
is most ethically appropriate when considering biosafety measures? How
can accidental introduction of synthetic life in the natural environment
be avoided? Much ethical consideration and critical thought has been
given to these questions. Biosafety not only refers to biological
containment; it also refers to strides taken to protect the public from
potentially hazardous biological agents. Even though such concerns are
important and remain unanswered, not all products of synthetic biology
present concern for biological safety or negative consequences for the
environment. It is argued that most synthetic technologies are benign
and are incapable of flourishing in the outside world due to their
"unnatural" characteristics as there is yet to be an example of a
transgenic microbe conferred with a fitness advantage in the wild.
In general, existing hazard controls, risk assessment methodologies, and regulations developed for traditional genetically modified organisms (GMOs) are considered to be sufficient for synthetic organisms. "Extrinsic" biocontainment methods in a laboratory context include physical containment through biosafety cabinets and gloveboxes, as well as personal protective equipment. In an agricultural context they include isolation distances and pollen barriers, similar to methods for biocontainment of GMOs.
Synthetic organisms may offer increased hazard control because they
can be engineered with "intrinsic" biocontainment methods that limit
their growth in an uncontained environment, or prevent horizontal gene transfer to natural organisms. Examples of intrinsic biocontainment include auxotrophy, biological kill switches, inability of the organism to replicate or to pass modified or synthetic genes to offspring, and the use of xenobiological organisms using alternative biochemistry, for example using artificial xeno nucleic acids (XNA) instead of DNA. Regarding auxotrophy, bacteria and yeast can be engineered to be unable to produce histidine,
an important amino acid for all life. Such organisms can thus only be
grown on histidine-rich media in laboratory conditions, nullifying fears
that they could spread into undesirable areas.
Biosecurity
Some
ethical issues relate to biosecurity, where biosynthetic technologies
could be deliberately used to cause harm to society and/or the
environment. Since synthetic biology raises ethical issues and
biosecurity issues, humanity must consider and plan on how to deal with
potentially harmful creations, and what kinds of ethical measures could
possibly be employed to deter nefarious biosynthetic technologies. With
the exception of regulating synthetic biology and biotechnology
companies, however, the issues are not seen as new because they were raised during the earlier recombinant DNA and genetically modified organism (GMO) debates and extensive regulations of genetic engineering and pathogen research are already in place in many jurisdictions.
European Union
The European Union-funded project SYNBIOSAFE
has issued reports on how to manage synthetic biology. A 2007 paper
identified key issues in safety, security, ethics and the
science-society interface, which the project defined as public education
and ongoing dialogue among scientists, businesses, government and
ethicists. The key security issues that SYNBIOSAFE identified involved engaging companies that sell synthetic DNA and the biohacking community of amateur biologists. Key ethical issues concerned the creation of new life forms.
A subsequent report focused on biosecurity, especially the so-called dual-use
challenge. For example, while synthetic biology may lead to more
efficient production of medical treatments, it may also lead to
synthesis or modification of harmful pathogens (e.g., smallpox).
The biohacking community remains a source of special concern, as the
distributed and diffuse nature of open-source biotechnology makes it
difficult to track, regulate or mitigate potential concerns over
biosafety and biosecurity.
COSY, another European initiative, focuses on public perception and communication. To better communicate synthetic biology and its societal ramifications to a broader public, COSY and SYNBIOSAFE published SYNBIOSAFE, a 38-minute documentary film, in October 2009.
The International Association Synthetic Biology has proposed self-regulation.
This proposes specific measures that the synthetic biology industry,
especially DNA synthesis companies, should implement. In 2007, a group
led by scientists from leading DNA-synthesis companies published a
"practical plan for developing an effective oversight framework for the
DNA-synthesis industry".
On July 9–10, 2009, the National Academies' Committee of Science,
Technology & Law convened a symposium on "Opportunities and
Challenges in the Emerging Field of Synthetic Biology".
After the publication of the first synthetic genome and the accompanying media coverage about "life" being created, President Barack Obama established the Presidential Commission for the Study of Bioethical Issues to study synthetic biology.
The commission convened a series of meetings, and issued a report in
December 2010 titled "New Directions: The Ethics of Synthetic Biology
and Emerging Technologies." The commission stated that "while Venter's
achievement marked a significant technical advance in demonstrating that
a relatively large genome could be accurately synthesized and
substituted for another, it did not amount to the “creation of life”.
It noted that synthetic biology is an emerging field, which creates
potential risks and rewards. The commission did not recommend policy or
oversight changes and called for continued funding of the research and
new funding for monitoring, study of emerging ethical issues and public
education.
Synthetic biology, as a major tool for biological advances,
results in the "potential for developing biological weapons, possible
unforeseen negative impacts on human health ... and any potential
environmental impact".
These security issues may be avoided by regulating industry uses of
biotechnology through policy legislation. Federal guidelines on genetic
manipulation are being proposed by "the President's Bioethics Commission
... in response to the announced creation of a self-replicating cell
from a chemically synthesized genome, put forward 18 recommendations not
only for regulating the science ... for educating the public".
Opposition
On March 13, 2012, over 100 environmental and civil society groups, including Friends of the Earth, the International Center for Technology Assessment and the ETC Group issued the manifesto The Principles for the Oversight of Synthetic Biology.
This manifesto calls for a worldwide moratorium on the release and
commercial use of synthetic organisms until more robust regulations and
rigorous biosafety measures are established. The groups specifically
call for an outright ban on the use of synthetic biology on the human genome or human microbiome. Richard Lewontin wrote that some of the safety tenets for oversight discussed in The Principles for the Oversight of Synthetic Biology
are reasonable, but that the main problem with the recommendations in
the manifesto is that "the public at large lacks the ability to enforce
any meaningful realization of those recommendations".
Health and safety
The hazards of synthetic biology include biosafety hazards to workers and the public, biosecurity
hazards stemming from deliberate engineering of organisms to cause
harm, and environmental hazards. The biosafety hazards are similar to
those for existing fields of biotechnology, mainly exposure to pathogens
and toxic chemicals, although novel synthetic organisms may have novel
risks. For biosecurity, there is concern that synthetic or redesigned organisms could theoretically be used for bioterrorism.
Potential risks include recreating known pathogens from scratch,
engineering existing pathogens to be more dangerous, and engineering
microbes to produce harmful biochemicals. Lastly, environmental hazards include adverse effects on biodiversity and ecosystem services, including potential changes to land use resulting from agricultural use of synthetic organisms.
Existing risk analysis systems for GMOs are generally considered
sufficient for synthetic organisms, although there may be difficulties
for an organism built "bottom-up" from individual genetic sequences.
Synthetic biology generally falls under existing regulations for GMOs
and biotechnology in general, and any regulations that exist for
downstream commercial products, although there are generally no
regulations in any jurisdiction that are specific to synthetic biology.
The central dogma of molecular biology is an explanation of
the flow of genetic information within a biological system. It is often
stated as "DNA makes RNA, and RNA makes protein", although this is not its original meaning. It was first stated by Francis Crick in 1957, then published in 1958:
The Central Dogma. This states that once "information" has passed into protein it cannot get out again. In more detail, the transfer of information from nucleic acid
to nucleic acid, or from nucleic acid to protein may be possible, but
transfer from protein to protein, or from protein to nucleic acid is
impossible. Information means here the precise determination of
sequence, either of bases in the nucleic acid or of amino acid residues
in the protein.
He re-stated it in a Nature paper published in 1970: "The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid."
Information flow in biological systems
A second version of the central dogma is popular but incorrect. This is the simplistic DNA → RNA → protein pathway published by James Watson in the first edition of The Molecular Biology of the Gene
(1965). Watson's version differs from Crick's because Watson describes a
two-step (DNA → RNA and RNA → protein) process as the central dogma. While the dogma, as originally stated by Crick, remains valid today, Watson's version does not.
The dogma is a framework for understanding the transfer of sequenceinformation between information-carrying biopolymers, in the most common or general case, in living organisms. There are 3 major classes of such biopolymers: DNA and RNA (both nucleic acids), and protein. There are 3 × 3 = 9
conceivable direct transfers of information that can occur between
these. The dogma classes these into 3 groups of 3: three general
transfers (believed to occur normally in most cells), three special
transfers (known to occur, but only under specific conditions in case of
some viruses or in a laboratory), and three unknown transfers (believed
never to occur). The general transfers describe the normal flow of
biological information: DNA can be copied to DNA (DNA replication), DNA information can be copied into mRNA (transcription), and proteins can be synthesized using the information in mRNA as a template (translation). The special transfers describe: RNA being copied from RNA (RNA replication), DNA being synthesised using an RNA template (reverse transcription), and proteins being synthesised directly from a DNA template without the use of mRNA. The unknown transfers describe: a protein being copied from a protein, synthesis of RNA using the primary structure
of a protein as a template, and DNA synthesis using the primary
structure of a protein as a template - these are not thought to
naturally occur.
Biological sequence information
The biopolymers that comprise DNA, RNA and (poly)peptides are linear polymers (i.e.: each monomer is connected to at most two other monomers). The sequence
of their monomers effectively encodes information. The transfers of
information described by the central dogma ideally are faithful, deterministic
transfers, wherein one biopolymer's sequence is used as a template for
the construction of another biopolymer with a sequence that is entirely
dependent on the original biopolymer's sequence. When DNA is transcribed
to RNA, it's complement is paired to it. DNA codes A, G, T, and C are
transferred to RNA codes U, C, A, and G, respectively. The encoding of
proteins is done in groups of three, known as codons according to the table.
DNA to RNA to Amino Acids
General transfers of biological sequential information
Table of the three classes of information transfer suggested by the dogma
General
Special
Unknown
DNA → DNA
RNA → DNA
protein → DNA
DNA → RNA
RNA → RNA
protein → RNA
RNA → protein
DNA → protein
protein → protein
DNA replications
In the sense that DNA replication must occur if genetic material is to be provided for the progeny of any cell, whether somatic or reproductive, the copying from DNA to RNA arguably is the fundamental step in the central dogma. A complex group of proteins called the replisome performs the replication of the information from the parent strand to the complementary daughter strand.
SSB protein that binds open the double-stranded DNA to prevent it from reassociating
RNA primase that adds a complementary RNA primer to each template strand as a starting point for replication
DNA polymerase III
that reads the existing template chain from its 3' end to its 5' end
and adds new complementary nucleotides from the 5' end to the 3' end of
the daughter chain
DNA polymerase I that removes the RNA primers and replaces them with DNA
This process typically takes place during S phase of the cell cycle.
Transcription
Transcription is the process by which the information contained in a
section of DNA is replicated in the form of a newly assembled piece of messenger RNA (mRNA). Enzymes facilitating the process include RNA polymerase and transcription factors. In eukaryotic cells the primary transcript is pre-mRNA. Pre-mRNA must be processed for translation to proceed. Processing includes the addition of a 5' cap and a poly-A tail to the pre-mRNA chain, followed by splicing. Alternative splicing
occurs when appropriate, increasing the diversity of the proteins that
any single mRNA can produce. The product of the entire transcription
process (that began with the production of the pre-mRNA chain) is a
mature mRNA chain.
Translation
The mature mRNA finds its way to a ribosome, where it gets translated. In prokaryotic
cells, which have no nuclear compartment, the processes of
transcription and translation may be linked together without clear
separation. In eukaryotic cells, the site of transcription (the cell nucleus) is usually separated from the site of translation (the cytoplasm),
so the mRNA must be transported out of the nucleus into the cytoplasm,
where it can be bound by ribosomes. The ribosome reads the mRNA triplet codons, usually beginning with an AUG (adenine−uracil−guanine), or initiator methionine codon downstream of the ribosome binding site. Complexes of initiation factors and elongation factors bring aminoacylatedtransfer RNAs
(tRNAs) into the ribosome-mRNA complex, matching the codon in the mRNA
to the anti-codon on the tRNA. Each tRNA bears the appropriate amino acid residue to add to the polypeptide
chain being synthesised. As the amino acids get linked into the growing
peptide chain, the chain begins folding into the correct conformation.
Translation ends with a stop codon which may be a UAA, UGA, or UAG triplet.
The mRNA does not contain all the information for specifying the
nature of the mature protein. The nascent polypeptide chain released
from the ribosome commonly requires additional processing before the
final product emerges. For one thing, the correct folding process is
complex and vitally important. For most proteins it requires other chaperone proteins
to control the form of the product. Some proteins then excise internal
segments from their own peptide chains, splicing the free ends that
border the gap; in such processes the inside "discarded" sections are
called inteins.
Other proteins must be split into multiple sections without splicing.
Some polypeptide chains need to be cross-linked, and others must be
attached to cofactors such as haem (heme) before they become functional.
Special transfers of biological sequential information
Reverse transcription
Unusual flows of information highlighted in green
Reverse transcription is the transfer of information from RNA to DNA
(the reverse of normal transcription). This is known to occur in the
case of retroviruses, such as HIV, as well as in eukaryotes, in the case of retrotransposons and telomere
synthesis.
It is the process by which genetic information from RNA gets transcribed
into new DNA. The family of enzymes that are involved in this process
is called Reverse Transcriptase.
RNA replication
RNA replication is the copying of one RNA to another. Many viruses
replicate this way. The enzymes that copy RNA to new RNA, called RNA-dependent RNA polymerases, are also found in many eukaryotes where they are involved in RNA silencing.
RNA editing, in which an RNA sequence is altered by a complex of proteins and a "guide RNA", could also be seen as an RNA-to-RNA transfer.
Direct translation from DNA to protein
Direct translation from DNA to protein has been demonstrated in a cell-free system (i.e. in a test tube), using extracts from E. coli
that contained ribosomes, but not intact cells. These cell fragments
could synthesize proteins from single-stranded DNA templates isolated
from other organisms (e,g., mouse or toad), and neomycin
was found to enhance this effect. However, it was unclear whether this
mechanism of translation corresponded specifically to the genetic code.
Transfers of information not explicitly covered in the theory
Post-translational modification
After protein amino acid sequences have been translated from nucleic
acid chains, they can be edited by appropriate enzymes. Although this is
a form of protein affecting protein sequence, not explicitly covered by
the central dogma, there are not many clear examples where the
associated concepts of the two fields have much to do with each other.
Inteins
An intein is a "parasitic" segment of a protein that is able to
excise itself from the chain of amino acids as they emerge from the
ribosome and rejoin the remaining portions with a peptide bond in such a
manner that the main protein "backbone" does not fall apart. This is a
case of a protein changing its own primary sequence from the sequence
originally encoded by the DNA of a gene. Additionally, most inteins
contain a homing endonuclease
or HEG domain which is capable of finding a copy of the parent gene
that does not include the intein nucleotide sequence. On contact with
the intein-free copy, the HEG domain initiates the DNA double-stranded break repair
mechanism. This process causes the intein sequence to be copied from
the original source gene to the intein-free gene. This is an example of
protein directly editing DNA sequence, as well as increasing the
sequence's heritable propagation.
Methylation
Variation in methylation states of DNA can alter gene expression levels significantly. Methylation variation usually occurs through the action of DNA methylases. When the change is heritable, it is considered epigenetic. When the change in information status is not heritable, it would be a somatic epitype.
The effective information content has been changed by means of the
actions of a protein or proteins on DNA, but the primary DNA sequence is
not altered.
Prions
Prions are proteins of particular amino acid sequences in particular conformations. They propagate themselves in host cells by making conformational changes
in other molecules of protein with the same amino acid sequence, but
with a different conformation that is functionally important or
detrimental to the organism. Once the protein has been transconformed to
the prion folding it changes function. In turn it can convey
information into new cells and reconfigure more functional molecules of
that sequence into the alternate prion form. In some types of prion in fungi this change is continuous and direct; the information flow is Protein → Protein.
Some scientists such as Alain E. Bussard and Eugene Koonin have argued that prion-mediated inheritance violates the central dogma of molecular biology. However, Rosalind Ridley in Molecular Pathology of the Prions
(2001) has written that "The prion hypothesis is not heretical to the
central dogma of molecular biology—that the information necessary to
manufacture proteins is encoded in the nucleotide sequence of nucleic
acid—because it does not claim that proteins replicate. Rather, it
claims that there is a source of information within protein molecules
that contributes to their biological function, and that this information
can be passed on to other molecules."
Natural genetic engineering
James A. Shapiro argues that a superset of these examples should be classified as natural genetic engineering
and are sufficient to falsify the central dogma. While Shapiro has
received a respectful hearing for his view, his critics have not been
convinced that his reading of the central dogma is in line with what
Crick intended.
"I called this idea the central dogma, for two reasons, I suspect. I had already used the obvious word hypothesis in the sequence hypothesis,
and in addition I wanted to suggest that this new assumption was more
central and more powerful. ... As it turned out, the use of the word
dogma caused almost more trouble than it was worth. Many years later Jacques Monod pointed out to me that I did not appear to understand the correct use of the word dogma, which is a belief that cannot be doubted. I did apprehend this in a vague sort of way but since I thought that all
religious beliefs were without foundation, I used the word the way I
myself thought about it, not as most of the world does, and simply
applied it to a grand hypothesis that, however plausible, had little
direct experimental support."
"My mind was, that a dogma was an idea for which there was no reasonable evidence. You see?!" And Crick gave a roar of delight. "I just didn't know what dogma meant.
And I could just as well have called it the 'Central Hypothesis,' or —
you know. Which is what I meant to say. Dogma was just a catch phrase."
Comparison with the Weismann barrier
In August Weismann's germ plasm theory, the hereditary material, the germ plasm, is confined to the gonads. Somatic cells (of the body) develop afresh in each generation from the germ plasm. Whatever may happen to those cells does not affect the next generation.
The Weismann barrier, proposed by August Weismann in 1892, distinguishes between the "immortal" germ cell lineages (the germ plasm) which produce gametes and the "disposable" somatic cells. Hereditary information moves only from germline cells to somatic cells
(that is, somatic mutations are not inherited). This, before the
discovery of the role or structure of DNA, does not predict the central
dogma, but does anticipate its gene-centric view of life, albeit in
non-molecular terms.
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