Synthetic Biology: Definitions, Concepts, and Applications

Posted on Aug 24, 2024 in Biology

Synthetic Biology (Definitions & Concepts)

Synthetic biology (interdisciplinary) is composed of biology, chemistry, engineering & refactoring.

Analogy to Organic Synthesis

Synthesis and analysis are complementary. In organic chemistry, analysis and synthesis were both critical in determining fundamental principles of chemical structure and reactivity. Synthetic molecules have been used for a wide variety of applications. Similarly, synthetic approaches will complement analytical methods in elucidating biological principles, and synthetic cellular systems will prove highly useful.

The Engineering Cycle in Synthetic Biology

Synthetic biologists apply the engineering cycle to biology:

  • Specification
  • Design
  • Modeling
  • Assembly
  • Testing

Design is a central issue of SynBio.

BioBricks: Standardized Biological Parts

BioBricks are standardized biological parts (e.g., promoters) that are:

  • Simple
  • Modular
  • Well-characterized
  • Standardized
  • Can be integrated into a cell “chassis” (aka “host”)

Synthetic biology parts, devices, and systems should be modular, fully characterized & standardized (many are not fully characterized). BioBricks are connected to devices & systems (which is not easy).

Computer-Aided Design (CAD) in Synthetic Biology

Systematic computer-aided (CAD) design of parts, devices & systems:

  • Block Diagram Design -> BioPart Block Diagram -> Assembly Strategy Software -> DNA Assembly -> Viability Testing (Wet Lab)

BioCAD: Biological Computer Aided Design

SynBIS: Synthetic Biology Information System

Synthetic Biology (Applied SynBio: Selected Examples)

Genome Assembly & Transplantation

Mycoplasma genitalium (with small modifications termed: M. genitalium JCVI-1.0)

  • Small parasitic bacterium
  • Lives on the ciliated epithelial cells of the primate genital & respiratory tracts
  • Smallest known genome that can constitute a cell
  • Circular chromosome of 582,970 bp
  • 521 genes
  • 482 protein-encoding genes
  • A minimal set of 382 genes can sustain life (Minimal Genome Project)

Minimal Genome Project (Mycoplasma laboratorium)

A strategy was proposed to synthesize a genome containing only the essential genes of a Mycoplasma species.

Watermarks

Watermarks are introduced to control (embedded messages within open reading frames of synthetic genes).

Genome Synthesis & Assembly

A plan for the five-stage assembly of the M. genitalium chromosome. In the first stage of assembly, four cassettes are joined to make an A-series assembly approximately 24 kb in length (assembly 37-41 contained five cassettes). In the next stage, three A-assemblies are joined together to make eight ~72-kb B-series assemblies (assembly B62-77 contained four A-series assemblies). The eighth genome B-assemblies are taken two at a time to make quarter-genome C-series assemblies. These assemblies were all made by in vitro recombination (A-series cassette assembly) and cloned into E. coli using BAC vectors.

Half-genome and whole-genome assemblies were made by in vivo yeast recombination. Assemblies in bold boxes were sequenced to verify their correctness. For the final molecule, the D-series half molecules were not employed. Rather, the whole molecule was assembled from the four C-series quarter molecules.

A-Series Cassette Assembly

Assembly of synthetic cassettes by in vitro recombination. It illustrates the reaction used for the first stage of assembly of the overlapping cassettes. Recombinant plasmids bearing the individual cassette DNA inserts were cleaved with the appropriate type IIS restriction enzymes, which cleave outside of their recognition site to the overlapping DNA molecules are digested with a 3’ exonuclease to expose the overlaps, (ii) the complementary overlaps are annealed, and (iii) the joints are repaired.

Polymerase chain reaction (PCR) amplification was used to produce a unique BAC vector for the cloning of each assembly, with terminal overlaps to the ends of the assembly. Each PCR primer includes an overlap with one end of the BAC, a NotI restriction site, and an overlap with one end of the cassette assembly. Cassettes were assembled, four at a time, in the presence of the appropriate BAC vector. Because the M. genitalium JCVI-1.0 genome does not contain a NotI site, all of the assemblies can be released intact from the BAC. For example, the assembly A66-69 was constructed by mixing together equimolar amounts of the four cassette DNAs and the linear PCR–amplified BAC vector specific for this assembly, BAC 66-69, as described above. The 3′ ends of the mixture of duplex vector and cassette DNAs were then digested to expose the overlap regions using T4 polymerase in the absence of 2′-deoxyribonucleoside-5′-triphosphates (dNTPs). The T4 polymerase was inactivated by incubation at 75°C, followed by slow cooling to anneal the complementary overlap regions. The annealed joints were repaired using Taq polymerase and Taq ligase at 45°C in the presence of all four dNTPs and nicotinamide adenine dinucleotide (NAD).

Higher Order Assemblies

Repair of annealed junctions containing nonhomologous 3′ and 5′ NotI sequences. The 3′ GC nucleotides are removed during the chew-back reaction. In the repair reaction, the 5′-GGCCGC NotI overhangs are removed by the 5′-exonuclease activity contained in the Taq polymerase.

Final Assembly – Homologous Recombination in Yeast

Yeast TAR cloning of the complete synthetic genome. (A) The vector used for TAR cloning contains both BAC and YAC sequences. Recombination of vector with insert occurs at “hooks” added to the TARBAC by PCR amplification. A yeast replication origin (ARS) allows for propagation of clones because no ARS-like sequences exist in the M. genitalium genome. Selection in yeast is by complementation of histidine auxotrophy in the host strain. BAC sequences allow for potential electroporation into E. coli of clones purified from yeast. (B) M. genitalium JCVI-1.0 quarter genomes were purified from E. coli, NotI–digested, and mixed with a TARBAC vector for cotransformation into S. cerevisiae, where recombination at overlaps from 60 to 264 bp combined the six fragments into a single clone. The TARBAC was inserted into the BsmBI site in C50-77.

Genome Transplantation (“Booting”)

  • The incoming genome must establish essential functions, such as replication, for its maintenance within a recipient cell.
  • Detailed mechanism of genome transplantation unknown
  • Temporary co-existence of the synthetic and natural genomes?
  • Transplantation ≈ two plasmids belonging to the same incompatibility group?
  • Two plasmids are incompatible when one destabilizes the inheritance of the other
  • Without a selection-marker, the one showing lower fitness will be lost during cell division

Things to Know About “Synthia”

  • “Synthetic genome” -> construction & information content
  • -> construction = all-synthetic
  • -> information content ≈ natural
  • DNA amplification on bacterial artificial chromosomes (BAC clones) in E. coli
  • Assembly of large DNA fragments by homologous recombination in yeast
  • Expression of M. mycoides genes in heterologous organisms most probably does not interfere with host functions
  • -> UGA (amber stop codon) encodes Trp in Mycoplasmas
  • -> most M. mycoides genes not expressed or as truncated proteins
  • Large DNA fragments are susceptible to physical fragmentation when handled in vitro (genome transplantation)

For the construction of the synthetic Mycoplasma genome, the DNA for the whole genome was synthesized as 50 nt oligo-nucleotides which were then assembled using gene synthesis, Gibson cloning & homologous recombination in yeast.

The synthetic Mycoplasma genome is synthetic in terms of construction, not information content.

Mycoplasmas use a different genetic code (UAG= Trp instead of STOP) which supposedly facilitates cloning in foreign hosts.

BAC = bacterial artificial chromosome

YAC = yeast artificial chromosome

Biosynthesis of Artemisininic Acid in Yeast

Artemisinin

  • Herbal drug
  • Secondary plant metabolite
  • Abundant in the leaves of Artemisia annua, the sweet wormwood
  • Discovered by systematic examination of plants used in traditional Chinese medicine
  • Sesquiterpene with unusual peroxide bridge (antimalarial effect)
  • The peroxide is responsible for the unique antimalarial action of the drug
  • Derivatives by chemical synthesis to improve solubility
  • Produced on a multi-ton scale from plants grown on fields in China & Vietnam

The flux through the yeast mevalonate pathway is improved by upregulating all relevant genes of this pathway.

The genes encoding enzymatic activities for the biosynthesis of amorphadiene, artemisinic alcohol, artemisinic aldehyde & artemisinic acid were imported from A. annua.

Amophadiene synthase, cytochrome P450 CYP71AV1 and its reductase CPR1 are expressed from a plasmid, all other genetic modifications were introduced into the yeast genome.

The biosynthesized artemisinic acid is chemically transformed into artemisinin.

ð Antimalarial drug!

Metagenomics, Microalgae & SynBio

Genomics & Metagenomics

Genomics: analysis of genomic DNA from an individual organism or cell.

Metagenomics: analysis of genomic DNA from a whole community.

Metagenomics Study Interests (Find New Metabolic Pathways or Motives for a Bioproduct)

  • Total extraction of the DNA from the samples (which you don’t cultivate): environmental single-gene surveys; shotgun studies of all environmental genes.
  • DNA sequencing: Identify common genes within a community; identify genome contents favored by current environmental conditions.
  • Protein annotation: use metagenomics studies as a tool to answer broader ecological or evolutionary questions.

Environmental Shotgun Sequencing (ESS)

  1. Sampling from habitat
  2. Filtering particles, typically by size
  3. DNA extraction and lysis
  4. Cloning and library
  5. Sequence the clones
  6. Sequence assembly

Metagenomics Issues

  • Direct next-generation sequencing of DNA without heterologous cloning
  • High-throughput computational techniques to cope with the analysis of millions of sequencing reads
  • Data analysis is the key limiting factor in metagenomic studies
  • Bioinformatics software and computational capacity are limiting
  • Sequencing costs drop much faster than computing costs
  • Standard formats for data sharing

Marine Metagenomics Key Data

  • Shotgun Sanger sequencing
  • 1 billion nonredundant base pairs
  • 1,800 unique genomes
  • 48 unknown bacterial phylotypes
  • 1.2 million previously unknown genes
  • GOS (Global Ocean Sampling): 6.3 billion base pairs from 7.7 million sequence-reads
  • Vastly uncharacterized metabolism
  • Increased complexity of biogeochemical pathways

Microalgae

  • Microscopic algae
  • Unicellular eukaryotes
  • Enormous biodiversity
  • Perform photosynthesis
  • Freshwater & marine systems
  • Exist individually, in chains, or groups
  • Pan-genome, e.g., Emiliana huxleyi
  • Metabolic repertoires dependent on the environment
  • (Secondary) metabolites in high concentrations (carotenoids, poly-unsaturated fatty acids, etc.)

Synthetic Biology in Microalgae

  • Designed algal biofuel production strains (biodiesel, H2, EtOH)
  • Improved terpenoid production; re-engineering for heterotrophism
  • Tools for genetic manipulation of several algal species
  • Six annotated chlorophyte genomes (Phytozome)
  • Algal metabolic models (BioModels Database)

Microalgae as Production Hosts (Challenges)

  • Photosynthetic quantum yield needs improvement
  • Currently, only microalgae products that have a very high value per kilo (e.g., carotenoids) are economic
  • Often sub-standard quality products
  • New technologies for massive algae culture & harvest needed (improved photobioreactors)
  • For economic biodiesel production, the price for microalgae production needs to be reduced substantially
  • The scale of lipid production needs to be noticeably elevated

ð Potential productivity of microalgae is 10-fold greater than that of agricultural crops & production could take place on non-arable land.

Conclusion

  • Metagenomics facilitates the discovery & study of new species, genomes & metabolic pathways.
  • Currently, data analysis is the key limiting factor in metagenomic studies.
  • The Global Ocean Sampling expedition collected a wealth of genetic information that is freely accessible via CAMERA.
  • Microalgae are regarded as the “plantimals” for future SynBIO applications.
  • The CAMERA database provides a framework to design and implement your own data analysis pipeline.