Synthetic Biology and the Creation of New Life Forms

The field has evolved from merely digitizing biology (like sequencing the human genome) to actively designing and synthesizing entirely new life forms with customized functions.
A fundamental, persistent question in this field is: What fundamentally defines life itself?

Mycoplasma genitalium possesses one of the smallest known genomes capable of self-replication in a laboratory setting, making it an ideal candidate for synthetic biology research.
The project's objective is to engineer an even more streamlined, minimal genome that retains only the essential genes necessary for survival and replication.
Researchers successfully inactivated approximately 100 genes from the organism's original set of around 500 genes to identify non-essential genes.
The metabolic map of Mycoplasma genitalium is relatively simple compared to more complex organisms, offering a more manageable platform for understanding basic life processes.
Experimentally knocking out single genes is typically insufficient to create a viable, living cell due to complex gene interactions and redundancies.
Synthesizing the entire chromosome is crucial for systematically varying genetic components and addressing fundamental inquiries into the nature and requirements of life.

A central challenge revolves around whether modern chemistry can facilitate the creation of large, complex DNA molecules.
Another key question is whether an inert, chemically synthesized chromosome can be "booted up" or activated within a living cell to take over cellular functions.
The pace of digitizing life, particularly in sequencing, has seen exponential growth, outpacing our capabilities in synthesizing genetic material.
Our ability to write genetic code, though increasing, is not advancing at the same rapid pace as our ability to read it, creating a bottleneck in synthetic biology.
The project was initiated more than 15 years ago, with initial phases focusing on thorough bioethical reviews and considerations.

Synthesizing DNA presents several technical difficulties. Most current machines can only produce short DNA fragments, typically ranging from 30 to 50 base pairs.
- These synthesis processes are inherently error-prone, with errors accumulating as sequence lengths increase, complicating the assembly of large DNA molecules.
To overcome these challenges, researchers developed a novel method to assemble small DNA fragments accurately and correct errors that arise during synthesis.

The project commenced with the digital information of the genome of phi x 174, a small virus that infects bacteria.
The designed DNA molecule, approximately 5,000 base pairs long, was inserted into bacteria.
The bacteria read the genetic code, producing viral particles that killed E. coli cells.
This part of the study served as a proof of concept, demonstrating that software (genetic code) could direct the construction of its own hardware (a biological system).
- Using a simple virus confirmed the ability to synthesize DNA and have it function within a living organism.

The main objective was to construct an entire bacterial chromosome, comprising over 580,000 base pairs and containing all the genetic information necessary for life.
The chromosome was assembled in modular units called cassettes (each comparable in size to a virus) to facilitate variation and enhance understanding of the essential components of a living cell.
Accurate design is essential because the digital information must be precise to ensure the synthetic chromosome functions correctly once introduced into a cell.
When the genome was initially sequenced in 1995, it had an error rate of 1 error per 10,000 base pairs. Re-sequencing identified 30 errors that would have prevented successful booting..
- To ensure accuracy, the design process involves overlapping 50-letter pieces to build smaller subunits, which are then pieced together.
Unique elements, including watermarks, were strategically designed into the synthetic chromosome using the genetic code to embed hidden messages or identifiers.
The chromosome pieces can be manipulated using enzymes, allowing for precise cutting, joining, and modification of the DNA sequences.
Pieces of increasing size were made: 5-7,000 letters, then 24,000 letters, and finally 72,000 letters.
Each stage involved growing the pieces in abundance for sequencing to ensure robustness and automation.

Large DNA pieces (over 100,000 base pairs) do not readily grow in E. coli, exhausting conventional molecular biology tools.
Homologous recombination, a natural DNA repair mechanism, was used to assemble large pieces in yeast.
An organism called Deinococcus radiodurans can withstand extreme radiation levels (3,000,000 rads).
The organism can repair its chromosome after it has been shattered by radiation.
The mechanisms used by organisms such as Deinococcus radiodurans suggest that life can exist, and move through space.
The synthesized molecule was so large it could be seen using a light microscope.
The final molecule contains over 580,000 letters of genetic code.
The Molecule has a molecular weight of over 300,000,000.

In eukaryotes (like humans), cloning is achieved by transferring a nucleus.
For bacteria and archaea, the chromosome is integrated into the cell.
A complete chromosome transplant from one cell to another can activate the new chromosome.
The chromosome from one microbial species was purified, and a few extra genes were added for selection purposes.
Enzymes were used to digest the proteins.
The chromosome was inserted into the cell. The new chromosome then replaced the existing chromosome.
Restriction enzymes in new chromosome digested the old chromosome.
The new chromosome took over, changing all the proteins and membranes, and converting the cell to the new species.
Reading the genetic code confirmed that it matched the transferred code.
Successfully moving DNA software can dramatically change organisms.

This work builds upon 3.5 billion years of evolution.
This could lead to a new Cambrian explosion, with massive speciation based on digital design.
This technology may help to provide resources for a global population of 9 billion people (up from 6.5 billion).
Currently, over 5,000,000,000 tons of coal and 30,000,000,000+ barrels of oil are used each year.
There is a need to replace such resources with biological processes, despite the challenges.
A database of approximately 20,000,000 genes is available, providing the design components of the future.
Combinatorial genomics techniques allow for the creation of a million chromosomes per day.
This enables optimization of processes such as the production of octane, pharmaceuticals, and new vaccines.
Design software is being developed to design species in the computer.
The focus is on next-generation fuels, moving beyond corn-to-ethanol.
Developments include sugar-to-high-value fuels, such as octane or butanol.
The ultimate goal is to use $CO<em>2$ as a feedstock, with sunlight and $CO</em>2$ as a method.
Organisms that convert $CO_2$ to methane, using molecular hydrogen as an energy source, are being investigated.
The aim is to capture $CO_2$ and convert it back into fuel.
The goal is to replace the petrochemical industry and become a major energy source.
The same tools are being used to create instant sets of vaccines.
The future may involve accelerated evolution through synthetic bacteria, archaea, and eukaryotes.