Synthetic biology

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The synthetic biology is a field in the border area of molecular biology , organic chemistry , engineering sciences , nano-biotechnology and information technology . It is described by some of its representatives as the latest development in modern biology .

In synthetic biology, biologists , chemists and engineers work together to create biological systems that do not occur in nature . The biologist thus becomes the designer of individual molecules, cells and organisms, with the aim of creating biological systems with new properties.

Various strategies are followed:

  • Artificial, biochemical systems are integrated into living beings , which thereby acquire new properties.
  • In accordance with the biological models, chemical systems are gradually built up in such a way that they have certain properties of living beings ( biomimetic chemistry).
  • Organisms are reduced to their most essential system components ( minimal genome ), which serve as a kind of “scaffolding” in order to create biological circuits by incorporating so-called bioparts .

In contrast to genetic engineering , not only z. B. individual genes are transferred from organism A to organism B, but the goal of synthetic biology is to create complete artificial biological systems . These systems are subject to evolution , but should be made "mutation-robust" to a certain extent.

History of Synthetic Biology

As early as 1912 the Frenchman Stéphane Leduc (1853–1939) published a work entitled “La Biologie Synthétique”, which dealt with the formation of plant-like forms when heavy metal salts are added to an aqueous solution of sodium silicate, a so-called chemical garden . In contrast, the chemist Emil Fischer formulated the program of a targeted chemical change of organisms for technical purposes at the end of the 19th century, for which he first used the expression "chemical-synthetic biology" in 1915.

In 1978 Waclaw Szybalski wrote in his comment on the Nobel Prize in Medicine for the work of Werner Arber , Daniel Nathans and Hamilton Smith on restriction enzymes : “ 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.

In 1980, the biologist and science journalist Barbara Hobom used the term to describe recombinant bacteria as a synonym for the application of genetic engineering methods.

In 2000, at the annual meeting of the American Chemical Society in San Francisco , Eric Kool described the integration of artificial chemical systems in living things as synthetic biology . and thus established the current understanding of this term.

In 2004 the first international scientific conference on synthetic biology took place at the Massachusetts Institute of Technology (MIT) in Cambridge, Massachusetts / USA, the SB 1.0 . This was followed by SB 2.0 (Berkeley, USA) in 2006 , SB 3.0 (Zurich, Switzerland) in 2007 and SB 4.0 (Hong Kong, China) in 2008 . The SB 5.0 took place in June 2011 on the premises of Stanford University in California, USA.

In 2007, researchers at the J. Craig Venter Institute (Rockville, Maryland) applied for patent protection in the USA for around 400 genes that are supposed to "operate" a bacterium constructed in the laboratory. According to the patent application, the bacterium's own genes should first be removed and then the genes compiled in the laboratory should be introduced with the possible aim of producing hydrogen or ethanol.

In January 2008, a research group led by Craig Venter reported that it had been possible for the first time to produce the genetic material of a bacterium completely synthetically. The model for the replica of the genome was Mycoplasma genitalium , Mycoplasma genitalium JCVI-1.0 was chosen as the name of the synthetic replica .

In May 2010, researchers at the J. Craig Venter Institute published that they had succeeded in inserting the complete, laboratory-synthesized genome of the bacterium Mycoplasma mycoides into a DNA-free cell from Mycoplasma capricolum . The synthetic genome encoded in the cell for proteins from Mycoplasma mycoides , so that the bacterial cells reproduced normally.

Construction of DNA

The modification of the construction principle of natural DNA and its behavior in bacteria led on the one hand to the revision of some model concepts and on the other hand resulted in a new diagnosis option for AIDS and hepatitis ( branched DNA diagnostic assay ).

A new backbone

In the 1980s, experiments were carried out with modified DNA in order to research the possibilities of specifically switching off genes with the help of antisense technology . Since the backbone of a polynucleotide strand consists of highly water-attracting ( hydrophilic ) building blocks ( phosphate and - in the case of DNA - deoxyribose sugar), these were replaced by fat-attracting ( lipophilic ) building blocks in order to be able to channel the molecules through the inner, lipophilic layer of the cell membrane . It was assumed that the modified backbone had no effect on the recognition of the complementary bases by the polymerases . The replacement of the backbone building blocks, however, led to many incorrect base pairings during the duplication ( replication ) of the DNA. The replacement of the nucleotides with PNAs ( polyamide-linked nucleic-acid analogues , molecules analogous to natural nucleic acids whose bases are linked by polyamides) resulted in chains that contained no more than 15 to 20 building blocks. Replacing the ribose with other, less hydrophilic molecules gave similar results.

However, nucleic acids with threose were more stable than their natural counterparts. In natural systems, this may not make sense, since too high a stability of the base pairing limits the possibilities for variation and thus the adaptability through evolution.

It has now been established that the phosphate-ribose scaffold plays an important role in the recognition system of the polymerases.

Extension of the genetic code

Hydrogen bonds of an adenine - thymine base pair
Hydrogen bonds of a guanine - cytosine base pair

The genetic code is based on four different bases, two of which are complementary to each other . Complementarity is based on the number and spatial orientation of the functional groups that enable the hydrogen bonds between the purine and pyrimidine bases . A hydrogen of the amino group (–NH 2 ) or the hydrogen of the nitrogen in the heterocycle acts as a donor (D), carbonyl oxygen or heterocyclic nitrogen with a non-bonding pair of electrons acts as an acceptor (A).

Theoretically there are 12 possible combinations for three hydrogen bonds for the combination of purines (pu) with complementary pyrimidines (py):

py pu py pu
AAD DDA DDA AAD
ADA (as with thymine ) DAD (as with adenine ) DAD ADA
ADD DAA DAA (as with cytosine , but here DA-) ADD (as with guanine )

This makes it possible to expand the genetic code by four new base pairs, which leads to 448 new codons . With a modified polymerase, a DNA that also contains these base pairs can be replicated.

Investigations with this modified replication system have shown that the DNA repair enzymes do not, as the previous model suggested, migrate along the small groove of the DNA double helix and search the nucleotide strand for non-binding electron pairs from the hydrogen bond donors.

Construction of enzymes

As early as 1983 Kevin Ulmer began to change the amino acid sequences of enzymes with the basic features of protein design in order to be able to generate new catalytic properties. The aim was to isolate certain domains that occur again and again in various enzymes, such as α-helix or β-sheet, as basic building blocks and to modify them in such a way that they can be combined as modules like a modular system to form various new enzymes with predetermined properties. Since the interactions, especially between more distant amino acid residues, are currently hardly known, this goal could not be achieved. However, in this research, enzymes were developed that are used as polymerases for DNA sequencing , as reverse transcriptases for the reproduction of artificial genetic systems, and as enzymes in detergents .

Construction of metabolic pathways

With the help of the recombination technology developed by Herbert W. Boyer and Stanley N. Cohen , which has been available since 1973 , transgenic organisms can synthesize certain substances that they would not have been able to produce with their natural genetic makeup . As a rule, a structural gene with the corresponding promoter and control genes is integrated into the host genome . With the help of the existing metabolic pathway , this gene is expressed and the desired substance is produced (example: synthesis of insulin by the bacterium Escherichia coli ).

Synthetic biology goes one step further in the construction of new metabolic pathways. The aim is a modular system of expressible genes that are assembled in a host organism as required and not only use the existing, natural metabolic pathways, but also establish new ones in the cell.

Since the numerous interactions between the numerous metabolic pathways and their regulation are only insufficiently known, a modular system has not yet been established.

Amorphadiene

However, the metabolic pathway from acetyl-CoA to amorphadiene could be established in an Escherichia coli strain . This substance is a precursor to artemisinin , which can be used as a drug against malaria . For this metabolic pathway, genes from the blood rain algae ( Haematococcus pluvialis ) and yeast ( Saccharomyces cerevisiae ) were used.

Artemisin is produced from the annual mugwort ( Artemisia annua ), but only in insufficient quantities.

Construction of biochemical signaling pathways

The basis is the regulation of gene expression , in which, for example, a signal molecule changes a protein molecule (repressor) that blocks transcription in such a way that it opens the way for the RNA polymerase and thus for the enzyme synthesis of a certain metabolic pathway. This metabolic pathway again produces molecules which in turn can serve as signal molecules for the induction or repression of the expression of certain genes.

The first combination of two different signaling pathways was achieved by linking two mechanisms that were not naturally dependent on one another: for example, a signal that actually promotes growth triggered cell death .

The approach of Tom Knight, Andrew (Drew) David Endy and Randy Rettberg (MIT Cambridge, USA) is the creation of modular genetic units, the so-called " BioBricks " ("biological building blocks"). These biobricks should, if they are in the genome z. B. be inserted by a bacterium, perform predefined tasks, analogous to the electronic circuits found in microprocessors (computers), hence the term "biological circuits". The idea of ​​Biobricks is that the Biobricks always fulfill a very specific task in the biological circuit, regardless of the (cellular or genetic) environment. However, it is not certain whether context independence can be adequately guaranteed in a complex biological circuit. Biobricks is still in an experimental phase.

Endy constructed a genetic circuit that made bacteria light up periodically. This so-called “repressilator” consists of three different BioBricks that inhibit each other. A building block also produces a fluorescent protein. The time delay causes an oscillation in this circuit:

Circuit of the repressor (R1, R2, R2 are the repressor molecules encoded by the corresponding genes, A is an activator molecule)

Although these genetic circuits imitate technical ones, there are fundamental differences that make technological application more difficult:

  • Genetic circuits are integrated into the genome of living things that reproduce and interact with the environment. The foreign genes are thus exposed to evolution. It was observed that after one hour, 58% of the manipulated cells no longer showed the desired behavior.
  • The building blocks represent foreign bodies in the host organisms. They remove nutrients from the cells for their function, so that the host's vitality is reduced.
  • The building blocks increase the complexity of the host cell. As a rule, this complexity does not allow exact predictions as in the corresponding simple technical systems based exclusively on the laws of nature.
  • In technical systems, signals can be brought specifically to their destination due to the wiring. In biological systems, a signaling molecule affects all system elements that have the corresponding receptor. So if the same modules are used in different parts of a circuit or in different circuits, there will be undesirable interactions. This problem is circumvented by the fact that different BioBricks have to be installed for the same function for each circuit in an organism. This leads to an undesirable increase in the number of modules. In some systems, the transcription rate of the modules (TIPS, transcription initializations per second ) is therefore used instead of a signal molecule .
  • The development of the "repressilator" took two years. It took James J. Collins one year to develop an E. coli strain with a flip-flop circuit.

Imitating electronic circuits initially seems like a gimmick. In addition to gaining knowledge about intracellular information channels, there are also opportunities for technological applications. It is conceivable that bacteria provided with appropriate genetic circuits indicate the presence of landmines, the signal being a certain concentration of TNT in the soil and the reaction being a light up in colors depending on the concentration of the TNT.

Complete genome synthesis

The first genome syntheses were based on fusion PCR , a method for generating genes from oligonucleotides .

  • In 2002 Jeronimo Cello, Aniko V. Paul and Eckard Wimmer put together infectious polioviruses in vitro .
  • In 2003 Hamilton O. Smith and Craig Venter succeeded in the complete synthesis of the bacteriophage PhiX174 with 5386 base pairs.
  • 2005: Partial synthesis of the influenza virus by Jeffery Taubenberger's group , which was responsible for the pandemic known as the Spanish flu around 1918 .
  • At the end of 2006 it was possible to produce synthetic DNA up to around 35,000 base pairs. Although the size of viable minimal organisms was estimated at only about 110,000 base pairs at the time, it was initially not possible to synthesize a complete prokaryote genome (bacterial genome).
  • In 2010, researchers working with Craig Venter announced the manufacture of the artificial bacterium Mycoplasma mycoides JCVI-syn1.0 . Previously, they had synthesized the 1.08 million base pair genome of a laboratory strain of Mycoplasma mycoides from chemical raw material and transferred it to a Mycoplasma capricolum bacterium that had previously been freed from DNA . According to the publication in Science , the cells equipped with the synthetic genome were found to be self-replicating and capable of exponential growth .
  • In March 2016, the team at the J. Craig Venter Institute in La Jolla, California presented the viable version Syn 3.0, which in turn is based on the bacterium Mycoplasma mycoides . The bioengineers shortened the original DNA molecule with 1079 kilobase pairs (kbp) to 531 kbp, thus reducing the gene repertoire by 57, namely to 473 genes. Surprisingly, the cellular function of 149 genes in the minimal genome remained unclear. So it remains to ask what constitutes life in molecular terms. Regardless of this fundamental problem, such a programmable minimal organism can be used for the industrial production of biomolecules.

Genome reconstruction

Endy and his team split the genome of the bacteriophage T7 into its known functional units, which were then pieced together again. Every combination was tested for its function. The hope was to distinguish the essential components from the superfluous, redundant ones and thus to find out which minimal genome is necessary for the transition from inanimate, chemical systems to living beings and thus to gain knowledge about the early evolution of living beings.

Experiments in silico

Capacities and computing power as well as special algorithms enable the simulation of complex biological networks in the computer. As a result, the methods in vivo (in the living organism) and in vitro (in the test tube) are expanded to include the option in silico (literally "in silicon"). With the computer, self-organization processes , such as the formation of membranes from amphiphilic molecules in water or the folding of proteins, can be examined and compared with observations made on natural systems. Another application is to investigate the importance of the base sequences of the human genome (see Human Genome Project ). Isolated base sequences of the DNA are translated into amino acid sequences with the help of the computer. These are compared with sequences of known proteins from model organisms such as fruit flies ( Drosophila melanogaster ), roundworms ( Caenorhabditis elegans ) or intestinal bacteria ( Escherichia coli ). On the basis of the protein structure found, the human protein molecule is constructed and examined in its spatial form in the computer. For example, active substances and drugs can be found and constructed on the computer that can influence the function of this protein.

A large increase in new questions, hypotheses and findings is expected from the simulation of regulatory networks of a cell in the computer. It has already been possible to reproduce the ventricular fibrillation of the heart in the computer using four genes that change their activity in the process.

Biosafety, ethics and intellectual property issues

In addition to the potentially positive uses of synthetic biology, the potentially negative effects must also be considered. In the USA and partly in Great Britain, the potential for abuse ( dual use ) by (bio) terrorists is being discussed in this regard, which is seen primarily in connection with the so-called war on terror . For example, the sequencing of pathogenic viruses (Spanish flu, polio) has led to a heated discussion as to whether and how the synthesis of pathogenic genomes can be restricted in order to prevent misuse. Warnings from some scientists, NGOs and journalists about bioterrorism raised awareness among the general public. An international association founded by gene synthesis and bioinformatics companies, the International Association of Synthetic Biology , laid the basis for a report with a detailed catalog of measures at a workshop on technical measures for ensuring biosafety in synthetic biology in Munich in 2008.

In Europe, future discussion is likely to be less about bioterrorists than about unintended health and environmental effects, ethics and intellectual property issues. Past and current public debates such as genetically modified seeds or stem cells show that new technologies can also contain potential for conflict.

Networking of synthetic biology in Germany

The German Association for Synthetic Biology e. V. (GASB), the largest relevant professional association in Germany, is dedicated to networking scientists and providing general information on the subject. In addition to scientific conferences, the association also organizes events for the general public.

Research activities in Germany are also promoted in the 'Synthetic Biology' study group of the Society for Biochemistry and Molecular Biology (GBM) , which enables the exchange of experts and young scientists.

literature

Press articles

Web links

Commons : Synthetic Biology  - collection of images, videos and audio files

multimedia

Individual evidence

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