“The chemically synthesised exact copy of the genome of an organism is called synthetic genome used to manipulate phenotypes or to create a new life form.”
the science of redesigning and synthesising entire DNA sequences of an organism is called synthetic genomics.
Designing and synthesising the entire genome is a very fascinating thing! But it is not as easy as its look.
Actually, we are here copying the genome or trying to write the genome as it is in the original form. We are not writing or designing a new DNA sequence.
It is just like replacing a car engine, instead of replacing a particular part, we are replacing our car engine to boost the performance, however, the body of the car is still the old one, we are inserting an engine that suite it.
Exactly, in the same manner, we are inserting a synthetic genome to our target cells. We are here trying to replace the native genome of the organism with the new one but not manipulating the pre-existing DNA sequences.
And that is the whole concept is all about. In the present article, we are going to discuss some of the interesting topics related to the synthetic genome and synthetic genomics.
“Re-designing, copying and synthesising the entire genome or all DNA sequences is called synthetic genome.”
“Artificially created genome used in order to create new life is called synthetic genome and the science is called synthetic genomics.”
What is a synthetic genome?
From the J. Craig Venture Institute, Rockville, Maryland, scientist Daniel Gibson and co-workers had first introduced the concept of a “synthetic genome.”
They had used three different species from which one’s genome was copied, assembled in another and expressed in some other organism.
They had selected Mycoplasma mycoides and synthesised 1.1 million base pair of its entire genome, assembled it in yeast cells and inserted it into the Mycoplasma capricolum.
In with it, to confirm the results they had inserted a fluorescent marker gene. After several cell divisions, blue coloured colonies were observed indicated the success of the experiment.
The artificially tailored genome of Mycoplasma mycoides was copied, some non-essential genes were removed and fever modification was made in the target organism to express the entire genome.
For example, a unique methylation pattern is one of the important property of any genome and that we can not create in a lab because you know, the addition of methyl group at each and ever exact position is nearly impossible.
For overcoming this problem scientists had removed the enzyme that degrades the unmethylation sequences.
What done by Gibson D and co-workers was truly incredible, and opened new doors for new genomic research.
But they have to thankful recombinant DNA technology for enabling them to do so.
The recombinant DNA technology was evolved during ‘70; restriction endonucleases, ligases and other essential tools and technique developed during this period.
Using the endonucleases and ligases the scientists were able to cleave and seal the DNA and thus artificial DNA can be constructed.
Restriction endonucleases: these are the enzymes that cut or digest the DNA sequence at its specific location called recognition site. The entire process is called restriction digestion.
Related article: Restriction digestion.
Ligase: ligase is the class of enzyme used to join or seal the DNA sequences, it forms phosphodiester bond between two adjacent nucleotides and seals the gap.
Related article: Ligase.
Methods for constructing a synthetic genome:
Polymerase cycling assembly:
The polymerase chain reaction is one of the best methods to do in vitro replication, it has the power to copy the entire strand of DNA or gene of our interest.
However, it can only amplify the DNA of several thousand base pairs.
Using the cycling assembly method combined with the polymerase chain reaction, we can synthesise a genome of more than several kilobase pairs, one of the example is Phi X 174 virus- the entire genome of this virus is synthesised using polymerase cycling assembling.
40 to 60 nucleotide long oligonucleotide complementary to our DNA are selected. The unique designing of the oligos unable the synthesis of an entire genome as the 20 nucleotide sequences on both ends of each oligo binds to two different complementary to the opposite strand.
In the very first step the denaturation is done which separates two DNA strands.
Hybridization of DNA sequences is done at 60°C followed by the polymerization through the Taq DNA polymerase.
All the gaps between all the DNA fragments are sealed by ligase.
Read more on PCR: Polymerase chain reaction.
TAR- transformation association recombination method is used to achieve homologous recombination between plasmid and genomic DNA.
The yeast artificial chromosome- YAC vector is constructed in such a way that behaves like the organisms own chromosome.
Using the polymerase chain reaction the gaps are repaired between the fragments utilising the extension primers.
(note: extension primers are the set of primers used to bind sequences flanking to our gene of interest).
The process of homologous recombination occurs between the YAC vector with the DNA cassettes and yeats own chromosome. A DNA cassette inserted into yeasts own chromosome. And replicates there.
Gibson assembly method:
The present method was originally developed by Gibson and co-workers and used in the synthesis of worlds first synthetic genome.
The method is a single-step isothermal reaction which synthesises larger DNA sequences than the polymerase cycling assembly method.
The isothermal reaction is performed followed by exonuclease and polymerisation.
The exonuclease creates an overhang complementary sequence on both ends and hybridized.
Then in the next step, the special type of DNA polymerase expands the DNA sequence.
In the final step, the ligase seals the gaps.
The Gibson assembly method can synthesise the genome more than 6Kb.
Rather to say fully functional technology, we can say, these are some of the trial and error methods to achieve artificial genome synthesis.
Because in 99% of the experiments Gibson and co-workers fail to achieve the synthesis.
Synthetic genome technology is used to create some of the economical important microbes such as biofuel secreting microbes, alcohol-producing microbes, for decontaminating toxic waste and to track down the tumour cells.
The entire process of constructing and introducing a synthetic genome is done is the five steps:
Writing or analysing the entire genomic DNA sequence of the target organism.
In the very first step, scientists must have to sequence the entire genome of an organism. Then the entire sequence is written in the computer and analysed and pre-process before synthesis.
Scientists have to understand the order and structure of the genomic sequences and how it is located on the chromosome.
Also, its hierarchy; its methylation pattern and exact location of essential genes.
Synthesising the genome using one of the methods enlisted above:
Synthesising the entire genome at once is not possible for now because we don’t have that much powerful technology, instead, fragments of an entire genome are created and each fragment is synthesised separately, in one or different laboratories.
For example, BioFab like groups is providing promoters and other regulatory genetic elements required to construct a synthetic genome.
BioFab currently has more than 350 promoters which are commercially available.
Assembling the genomic fragments:
Now in the next step, another tedious job is to assemble all the fragments orderly to construct the exact copy of the synthetic genome.
Ligating each and every fragment artificially is another nearly impossible job, for that scientists are using other organisms such as yeat.
Transplanting the synthetic genome in the target organism:
Once the synthetic genome is ready, it is transplanted into the target organism by removing the native genome with the synthetic one.
Boot up the cell to express the genomic content:
“Booting up” our synthetic genome is also one of the challenges as we don’t know more about how the entire mechanism is actually working.
Scientists must have to do some modifications to achieve the expression, for example, removing enzymes that cleave the unmethylated DNA sequences or removing those sequences which are not essential.
Application of synthetic genome:
The artificial genome synthesis can be beneficial in many ways, however, Gibson and c-workers (original developers of the concept) are still not clear about how it is useful to us.
Nonetheless, some of the possible applications of it are enlisted here.
Using synthetic genome technology, the newer biosynthetic pathway can be incorporated in microorganisms to produce biofuels, alcohol and detox agents.
Further to this, humanized antibodies can be generated artificially.
Unnecessary genomic sequences can be removed thus synthetic cells with minimal genome can be designed.
Humanized pig or animal model can be developed to create artificial organs which can solve the problem of transplantation failure.
Newer metabolic pathways can be inserted in the target organism to fulfil new functional requirements.
Other than this, it might have some therapeutic applications for human health.
However, these are some of the possible uses, none of the synthetic genome models is now fully available for therapeutic uses yet.
Limitations of the synthetic genome:
Developing an entire synthetic genome itself is a challenging task. Tedious and time-consuming wet and dry lab work are required in it.
An entire single genome can not be synthesised in a single lab, we have to assign work to different labs for creating different libraries of the genome, thus the chance of error is very high.
The overall cost of the project is yet another factor which is almost 10 times more than the gene therapy.
Booting up the artificially synthesised genome is another major problem, scientists still do not have knowledge about how exactly cell machinery boost up.
In addition to this, scientists have to modify a cell to adopt a new genome, even though it is exactly same as its native one, the chance of rejection is very high.
None of the present technology can synthesise exactly the same genome or an entire genome.
Even though, some of the bacterial and viral genomes are synthesised artificially, designing an entire genome of a eukaryote is just like a dream for now.
Synthetic genome is actually a fascinating and interesting concept but for now, it is impossible to commercialize it. The process is time-consuming, the success rate is unpredictable, the cost is very high, the process is uncertain and the outcomes are unknown.
We, scientists have to work had to make it real and applicable to mankind.
Baker, M. The next step for the synthetic genome. Nature 473, 403–408 (2011).