Biotechnolgy Handbook - Published by Butterworth/Ann Arbour Edited by Paul N.Cheremisinoff and Robert P. Ouelette - February 17, 1984
THE EMERGENCE OF BIOTICS
Predicting the future is a difficult task, especially in the field of biology. Technological innovations are generally the result of the convergence of several independent paths. This was the case with genetic engineering. It is because of dramatic advances in virology (the study of bacteriophages), bacteriology (the study of the colon bacillus and plasmids), molecular biology of the gene (the genetic code), and enzymology (restriction enzymes) that it was possible to gather the knowledge, methods, and techniques which built up this new field of research.
During the next decade, "classical" biotechnology will continue to develop in an important and spectacular manner. Genetic engineering will benefit from new transfer and cloning techniques. Eucaryotic cell cultivation will be improved through the extensive use of synthetic growth media and a better understanding of cellular growth factors. Gene transfer and gene expression in these cells, by means of multiorganism vectors, will be widely used. Hybridoma techniques will also experience considerable development in diagnostic tests, as in the development of therapeutic agents which can be specifically aimed at target cells. Molecular hybridization probes will be used to sort and select genes, for diagnostic or production purposes. Mammalian cloning techniques will be implemented by cattle breeders, and for mass production of laboratory animals. Enzymatic engineering will be applied to an increasing number of industrial processes, either biochemical (isomerizations, specific electrodes) or chemical (hemisyntheses). Considerable developments will occur in plant gene transfers. Such developments and applications are thoroughly described in other sections of this Handbook. This outstanding development of biotechnology during the last ten years has been caused principally by man's capability to decipher the codes of life, reprogram the cells, amplify the control signals by tuning appropriate molecular switches, and use adapted hosts capable to grow in large quantities. Most of this development has been centred around genetic engineering and hybridomas. Little is said generally about biotechnology equipment and machines which provide the environment on which new bodies of knowledge can grow, and which has a catalytic effect on the development of biology in all disciplines. New laboratory equipment and production apparatus has recently been born from the convergence of independent research and development paths in physics (mostly electronics, microelectronics, optics), organic chemistry (synthesis and analysis of macro-molecules), analytical chemistry, electro chemistry, immunology (antibody/antigene complex and detection), computer sciences.
In the early fifties and more recently, fields of interest were born from cross-fertilization between biology and physics: bionics, bio-electronics, biomimetics. It seems fruitful to bring together such fields of research by creating a new concept resulting from the convergence of several disciplines (as well as automatic engineering, data processing, and micro-electronics), with biology. A new concept capable of dealing with such various fields as: computers in biology; micro-electronics and new devices; biochemical reactions in automatic machines and analytical apparatus; electronic properties of molecules to make transducers, probes, chips; enzymes as micromachines and micro-robots; new biomaterials. It seems appropriate at this time to introduce a new word necessary to identify and recognize this hybrid new research field, at the heart of the development of biotechnology for the next 50 years. By analogy to other new fields related to computer science, such as telematics or robotics, I have proposed to name it "biotics" 1 -21 .
Biotics is a new field of research, development, and applications resulting from the marriage of severaldisciplines and particularly: solid state physics, micro-electronics, organic chemistry, electrochemistry,molecular biology, and through the extensive use of in-formation technology (data processing, automation,robots) in all areas of biology. This marriage has been made possible because of the following properties ofbiological and information systems:
1. The biological revolution which we have experienced for nearly 20 years, is in fact a revolution in the understanding of codes, languages, memories, and biological communication. Notions such as the genetic code, the control of gene expression, receptors, peptides hormones, or histocompatibility antigens, represent some of the elements of communication and network regulation of living systems. Therefore bridges can be built between biological systems and information science and engineering.
2. Biological informational macromolecules (DNA and proteins), are prone to the processing and memorization of data by computers. Biological macromolecules carry their information in a sequential and linear form. The sequential aspect of biological macromolecules means they can be treated like "printed text" on computers, automating the successive steps of their synthesis or analysis by a step by step identification or addition of the different nucleotides or amino acid sequences.
3. The last major reason which facilitates the convergence of biology and information sciences is our recent capability to handle molecules one by one and assemble them in any possible order. Before such micro-engineering techniques, we were only able to handle molecules, not as independent units, but statistically . Micro-electronics evolved from the macro, down to the micro, by minaturising electronic circuitry. For the first time it is now possible to reverse this classical engineering approach, and to work from the micro up to the macro, by taking advantage of the natural laws of atomic and molecular systems to self-assemble in three dimensional macro-molecular structures, and then in macromolecular arrays or sub-assemblies, which could then perform several dedicated functions at our level, acting like molecular machines.
THE BASIS OF BIOTICS: MASTERINC THE LANGUAGE OF LIFE THROUGH AUTOMATION AND COMPUTERS
To get a better evaluation of the full impact of biotics on biology and biotechnology, it is necessary to review some historical data. The advent of biotics was made possible in recent years by a better understanding of the language of life and through progresses in information sciences. The capability to decode, read, and write the language of life is turning a new page in the history of mankind, much in the same way as did the discovery of writing and printing centuries ago.
In 1953, two great hopes of biologists in their unending pursuit to understand life, were achieved—the complete analysis of a protein (insulin) by Frederick Sanger and the structure of the double helix by J. Watson and F. H. C. Crick . Then, Marshall Niremberg in 1963 deciphered the genetic code and discovered its similarity among bacteria and man . For the first time, molecular biologists were able to understand the meaning of the hereditary information, and establish the relationship between the genetic code, and the succession of amino acids in a protein sequence. The second step was accomplished during the 60s. The automation of the analyses became possible withthe improvement of Sanger's techniques and Pehr Edman's methods of protein degradation . The "sequantor," an automated machine for carrying out sequential degradation of peptides and proteins, was developed and commercialized. Sequences are now stored in computers in the form of "protein atlas" .
In 1976, Walter Gilbert and Alan M. Maxam of Harvard University  and Frederick Sanger , proposed a new rapid method of chemical analysis of DNA, allowing to read one by one the nucleic acids building blocks. The present challenge is the development of an automatic machine able to "read" the nucleic acid sequences. A Japanese company, Seiko, has recently proposed a microchemical robot able to perform such functions .
For reading protein sequences, a microanalyser, developed by Leroy Hood and Michael Huckapiller in California [12-13], allows to sequence 40 to 200 amino acids per day from only 10 nanograms of protein.
The first automatic protein synthesizing machines were developed and commercialized thanks to the work of R. B. Merrifield . They are presently used in many university research laboratories, and in the pharmaceutical industry. For nucleic acids, an important breakthrough occurred with the first synthesis of a gene by H. Gobind Khorana at the University of Wisconsin in 1964 [15-16], then at M.l.T.; Khorana's method was further improved by K. Itakura, of City of Hope Medical Center in California, who used it in 1978 and 1979 for the partial synthesis of the insulin and human growth hormone genes . These synthetic genes were introduced into bacteria by H. Boyer of Genentech [18-19] and expressed like natural genes with the aid of genetic engineering techniques.
In 1980, Vega Labs in California announced the first automatic gene machine. The machine had been developed by Itakura. Shortly after, a Toronto based company, BioLogicals, marketed a machine developed by Kevin Ogilvie of McGill University. In earIy 1981, Leroy Hood, the inventor of the microanalyser, in association with Genetic Instruments, proposed a new automatic gene machine . Several gene machines are now commercialized by a dozen biotechnology companies.
Now that we are partially mastering the codes and languages of life, and that we know how to produce them at will, where is this extraordinary evolution of techniques leading us to? Can we expect one day a library containing all the genetic information necessary to recreate a human being or functional parts of living systems? Whatever the answers to these questions might be, gene and protein synthesizing machines are already scientifically invaluable. Today, it is possible to produce any sequence of a gene. This synthesis is still being made step by step, but soon biologists will use prefabricated sequenccs which the machine will automatically call up and link to the other elements that enter into the synthetic gene. Computerized handling of such biological information constitutes one of the major impacts of biotics on many fields of biology and biotechnology, particularly in three major areas: gene libraries, analysis of sequence data, and molecular programming languages.
• The number of genetic messages deciphered and read in research laboratories throughout the world increases at the rate of 15 percent per month. This leads to the quadrupling of the known number of gene sequences in less than one year. The world ATLAS of protein sequences, had already stored over 400,000 gene sequences taken from international scientific publications 121-22]. Other gene libraries are implemented. They contain mainly natural gene sequences of viruses, bacteria, and human genes. Soon, with the aid of synthetic genes, there will probably be genetic libraries "subroutines," like in computer science, cataloging repetitive sequences which are common to several species of living organisms. Some companies specializing in genetic engineering equipment already manufacture on request such genetic subroutines including sequences of "promoters," "linkers," hybridization "probes," "start signals," needed by the cell machinery to control specific functions.
• Because of the sequential nature of nucleic acids and proteins, it is possible to compare and match sequences that have been stored in computers much as we compare sentences or words in our spoken languages. These analysis and matching procedures open the way to the equivalent of applications of modern word processing: searching for key words (i.e. sequences), coding data, storing information in data banks, finding homologies of sequences, etc. This capacity will allow to check if proteins with different properties are coded by similar genes; or find in different proteins a common antigenic sequence, likely to serve as a basis for the fabrication of a synthetic vaccine 123]. Recently, computer analysis of genetic sequences of a platelet factor involved in wound healing (PDGF), and of a simian sarcoma virus (SIS), has revealed interesting similarities, establishing important relationships between and oncogene and a naturally occuring factor l24-25].
• Computeri7ed sequence analysis can also help flnd "common ancestors" of certain proteins, thus extending the genealogical tree of living species to the level of molecular evolution. Genetic "word processing" can also help to determine automatically the restriction sites of the DNA molecule. Approximately 100 types of restriction enzymes are known today. Each one breaks DNA in precise places. The computer can indicate which fragment will be obtained if one enzyme is used rather than another. It can then contribute to gene splicing and editing routines by keeping track of the different restriction sequences and their specific ends (sticky ends). New stand-alone units, combining a microcomputer and graphic display capabilities have been recently introduced on the market to provide molecular biologists with tools for the planning and execution of gene splicing experiments. One of those units, called "BION" has been introduced by Intelligenetics [261. The future of such applications lies in the combination of large computers with numerical capabilities, sophisticated graphic displays, and artificial intelligence. Different systems can be linked through high-speed data transmission networks, like the Xerox Ethernet.
Gene synthesizing machines open the way for "gene programming machines" capable of writing programs which determine and control the fundamental reactions of life. A computer program consists of a succession of instructions stored in a memory, and executed one by one by the central processor. These instructions are organized in "words" holding the operational code and address where data must be stored and retrieved. All computers, micro, mini, or maxi operate on a limited number of instructions such as READ, PRINT, ADD, SUBTRACT, GOTO (unconditional branch), IFTHEN (conditional branch), SHIFT, START, STOP. These instructions are expressed by the user in high level languages, such as FORTRAN or BASIC. This high level language is then translated into a series of electronic impulses of binary digits 0 and 1, the only code understood by the computer.
Life programs are also written in a language common to all living beings. As the computer uses binary digits 0 and I, living systems use the four chemical letters of the genetic code: A, T, G, C. These bases, assembled three by three form words, the "codons." Each codon specifies an amino acid position in the protein chain, or other instructions necessary for the expression of the genetic program. The genetic code is read, sequence by sequence, by the enzyme RNA polymerase which transcribes DNA's information in copies of messenger RNA. Molecular biologists and geneticists know the codons corresponding to the start signal (INITIATION), and, to a certain extent, the stop sequences (TERMINATION), and punctuation. The binding sites of the repressor, a protein controlling gene expression, have also been identified as well as the sequences of the promoter gene on which the RNA polymerase is attached. One specific sequence appears to play an important role in initiation at this level, the code TATA (TATA BOX) [27-28] .
Our capacity of using an interdependent set of biotechnological tools, makes it possible to adopt a new integrated strategy for research and development in biotechnology: Computers are used to store and analyse biological sequence data; organic chemistry, for the synthesis of proteins and polynucleotides; small oligonucleotides, as probes to trap larger molecules through hybridation techniques  (RNAs or DNAs which could then be used for cloning experiments); peptides to produce monoclonal antibodies for the isolation of proteins [301. The coming years will see a tremendous improvement of such strategy, which is summarized by the following diagram. It shows clearly that any major progress in the biotechnology field, is now dependent on progress made in automatic macromolecular analysing and synthesizing machines, as well as in large computers for storing data, representing and handling macromolecular structures, or small desk-top computers to plan for cloning and other molecular biology experiments.
BIOMOLECULAR PROGRAMMING AND MICROBIOMACHINES
Today geneticists are in a position comparable to the one of computer specialists writing programs in binary language. They are at the stage computer science was during the earIy 50s. But already the codes and symbols they use lead them to go one step further: to the equivalent of "an assembly language." The terms they use (operons, introns, exons, transposons), represent a coded language. The next step will probably be the creation of a molecular programming language made possible by the development of gene synthesizing machines, and particularly by the progress made in the automatic assembly of sequences from previously stored data. Biologists will probably never reach an equivalent level of sophisticated languages like FORTRAN or BASIC, because the mechanisms of reading, assembly, and execution in biological systems are so different than those occurring in electronic computers. Nonetheless, in combining certain sequences, biologists already know how to write molecular programs with "loops," a repeated transcription of the same sequences (for instance, in multiple copy and thermosensitive plasmids). Will molecular programmers be able to perform "jumps" analogous to the GOTO instruction, from one sequence to another, or even conditional branching? Or more, bring in subroutines to help complete the molecular program? The advent of gene machines and genetic engineering techniques is opening the way toward a novel form of molecular programming language.
If such a potential was to be achieved, biotics would follow a path similar to that of editing in book and magazine publishing. We can expect a similar procedure with gene synthesizing machines or for molecular programming. The user will compose and complexify the programs of life, calling up entire pre-assembled sequences, and different modules of amplification and control.
Molecular programming languages will become even more important through the use of dedicated small computers linked to graphic displays, which will allow the molecular biologist to plan for cloning experiments. Coupled with powerful computer graphics programs for molecular design [31-32], this technology represents a new symbiosis between man and computers, enabling biologists and organic chemists to design at will any three-dimensional structure capable to perform dedicated functions . It is the opening of a new era: the micro-engineering of molecular machines.
But a critical factor for the future of biotics remains: once the program exists, how can it be expressed? By the use of what kind of machine? How will the "biosoftware" and the "biohardware" be combined? In computer sicence, the program is run by the computer; in genetic engineering, by living cells—bacteria or eucaryotic cells. Would it be possible to run a genetic prograrm by an "in vitro" system containing all the cellular elements necessary to read and run such a program? Molecular biologists are already using such "microbiomachines," the cell-free systems. These systems are made up of cellular extracts containing all the necessary elements for the synthesis of proteins (ribosomes, enzymes, nucleotides, transfer RNA molecules, mineral salts, ATP, etc.). When the genetic program, in the form of messenger RNA, is introduced into these systems, small quantities of the corresponding protein are immediately synthesized, although the yields are still quite low. In addition, these systems need energy in form readily usable by the enzymes. Research is continuing to improve yields and to insure the continuous supply of energy for biosynthesis. A possible solution would be the utilization of plant chloroplasts membranes (thylakoids). These thylakoids capture solar energy and transform it into chemical energy which in turn can be used by the microbiomachines.
The original aspect of using these automated microbiomachines is their possible operation with artificial gene programs (synthetic messenger RNA and transfer RNA molecules). Transfer RNA plays the role of an "adaptor" between messenger RNA codons, and the protein chain assembled on the ribosomes. The adaptor reads the messenger RNA code and positions the amino acid it carries, into its designated position in the protein chain. Theoretically, any artificial amino acid can be inserted in a protein chain. The contribution of organic chemists in this particular area is going to be very important. At the present time, they are able to synthesize "custom made" amino acids not found in nature, but having well defined physical and chemical properties. These synthetic amino acids could then be linked to artificial transfer RNA molecules and incorporated into artificial enzymes of predesigned properties, by a cellfree system of microbiomachines. These artificial proteins could lead to numerous applications: catalytic microsurfaces, specific electrodes, ultramicrocircuits, energy conversion microsystems.
Microbiomachines are already in use in all living systems. Proteins and enzymes are molecular machines, able to turn shafts, transmit tension, connect parts, move molecules, hold fluids, modify workpieces, move components. They are called flagellar motor, microtubules, ribosomes, collagen, vesicles, metallic complex, functional groups 13]. They can be used on production lines, with enzyme pools to construct complex macromolecular structures and assemblies. They can store and read programs like numerical control systems, as in the case of RNA and DNA reading and transcribing the genetic code. Cables, glue, containers, drive-shafts, motors, pipes, pumps, conveyor belts, already exist in the molecular world. Now with the knowledge gained in such fields as microbiology, enzymology, genetic engineering, and computer assisted molecular design, it is possible to engineer such micromachines, and assemble them on catalytic surfaces or arrays, transforming the micro-engineering capabilities of such machines into the macro-engineering necessities for mankind. Structural proteins and artificial enzymes will be used for the creation of ultrastructures analogous to those observed in the living cells. The properties of such artificial ultrastructure will be determined by the preferential orientation of the macromolecules in space, and their specific functions. Biologists will be able to reproduce the electron transport chains, like the cytochromes systems; the enzyme pools located side by side in the mitochondrial membranes; or the molecular units catalyzing the photosynthetic reactions in the chloroplasts membranes.
MOLECULAR INFORMATION PROCESSINC AND MOLECULAR ELECI'RONIC DEVICES
One of the most promising areas of biotics results from the convergence of microelectronics, solid state physics, and molecular biology . This hybrid technology is leading to the development of molecular electron devices (MED). Technological breakthroughs in this area are opening the way to the development of "chemical computers." Such long term applications were discussed during several meetings and workshops recently held in the United States [35,36,37]. Molecular electron devices could represent a significant breakthrough for computers in the next 30 years [38-39]. From the tube era (40s to 60s), to the transistor era (60s to 80s), we are now entering into the molecular electron devices era (80s to 2000 and beyond). The challenge is enormous. We need to start from scratch and reinvent all the components of present day micro-electronics .
First, engineers have to work on a switch, able to shift information in one state or another. By interrogating this component, one should be able to fnd out in which state the switch is. The development of a molecular switch will be equivalent to the discovery of the transistor.
The second major achievement will be the building of a true memory with different switches, that is, an array of molecules which could undergo reversible alterations, and therefore can be reused.
The third major step is to construct molecular wires able to transport information through distances, like conjugated chains of carbon atoms on which solitons move rapidly [41-421 in order to connect switches and memories.
The fourth step will be represented by the assembly of switches, memories and wires in three-dimensional structures or arrays, organized in assemblies of modules at several levels of communication and interconnections, able to perform coordinated functions. This is where one of the major breakthroughs of the new molecular engineering techniques will come into play: from the properties of atoms and molecules, the system will selfassemble in highly organized structures, putting atoms, molecules, and macromolecules into place, in order to perform dedicated functions. At this step, immunology techniques could be used to fit molecules at predetermined spots, or to deposit heavy metals which could be used as conductors at other levels of communications .
Finally, the system should be reparable. Modules which do not perform correctly will have to be detected, corrections made and sometimes a device replaced. These types of seif-repairing automata already exist in biological systems. We begin to understand how they function at the molecular level, and how they selfassemble. Other supramolecular assemblies, like the quantosome, the photosome, the oxysome are small self-contained factories which perform important functions for the cell. These minute devices are packed with an enormous amount of molecules and information. We are slowly starting to understand their structures, and able for the first time to copy them. But we still have to learn more from nature's design, understand how nature operates, and then translate what we have learnt into the new micromachines.
In order to make ultramicrocircuits, several laboratories are trying to synthesize proteins which do not exist in nature. Such syntheses are feasible using present day genetic engineering techniques, automatic gene and protein synthesis, and computer reproductions of the bi and tridimensional structures of amino acid sequences. It is likely that from this research a large variety of molecular electronic devices and ultramicrocircuits could be conceived and manufactured. They could be rendered biocompatible, thus allowing the production of implantable logic circuits, offering the prospect of direct interface between the central nervous system of animals or human beings and computers. Such biocompatible circuits are now being implanted and tested in the brains of rats [441. Other applications of these ultramicrocircuits could be the production of prostheses for the blind, different transducers, or solar energy converters on soft plastic sheets. A general preparation technique of biomolecular ultramicrocircuits has been patented in 1978 by EMV, a small company of the Washington area 145]. The production and assembly of these molecular circuits can be considered from two different approaches: a "passive" one (successive depositions, etching, grafting, doping) using technologies close to those presently used in the manufacturing of microcircuits; or an "active" one resulting from the spontaneous "growth" of the molecular circuit. In fact, automatic machines used today for synthesizing and analyzing genes and proteins, offer new models which may inspire the automation of the successive operations of molecular circuit production: growth of polymers, successive washing, reactions with other active groups, blocking, and reactivation of chemical groups.
However, at the level where the presence or absence of a single chemical link can affect the performance of an entire circuit, it is virtually impossible to construct and assemble circuits with traditional macroscopic control techniques. It bccomes necessary to use autoassembly properties of biological macromolecules, observed for instance in Langmuir-Blodgett films , or during the auto-organization of viruses or predissociated cellular organelles. In other words, instead of introducing the information from the outside, as we do today with most of our machinery (drill press, lathe, or even car construction robots), we will use information from the biopolymers themselves. Such information stored in the primary sequenees of amino acids, allows the three dimensional folding of a protein. It is thus possible to benefit from the properties of biological macromolecules to assemble three dimensional molecular ultramicroeircuits.
Many questions still remain unanswered: will these ultramicroeireuits be repairable? Will we be able to seleetively break chemical linkages or rearrange them? Is it still necessary to utilize boolean logic, presently used in all computers? Will we be inspired by the neuronal networks of the brain? These eireuits presently work, in all probability, in a non-boolean fashion, using parallel processing. To build the logie of the future, the convergence between molecular neurobiology and microelectronics is in the forefront and it holds great promise. From now on the evolution of bioties seems to be irreversible. In the next 50 years, this evolution will perhaps lead us to a "symbiotie" man, directly eonneeted by his own nervous system to miniature eomputers, able to tap through eommunication networks into any giant memory from any place on earth, or to communieate directly with any individual. This man/ computer interface will probably be achieved by logic circuits compatible with living tissues and operating at the request of an internal eommand. The molecular ultramicrocircuits open up the way towards "artificial senses" allowing, for example, to "see" in the infrared, to detect minute quantities of radioactivity or to enhance the capacities of recognition of certain odors. While we already have great difficulty in mastering our own neuronal circuits and effectively communicating with others, is it necessary to embark upon amplification techniques of our own intellectual and sensorial capacities vvith the aid of a controlled symbiosis with 1 machines? What will happen to our relations vvith the outside world? Will the "symbiotic man," related physically and biotechnologically to machines that he himself created, constitute a distinct living species, eventually replacing Homo sapiens? No one knows. But it is hard to satisfy the curiosity of scientists; especially when they discovcr at the intersection of several disciplines new unknown temtories to explore and possibly conquer....
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Edited by Paul N.Cheremisinoff and Robert P. Ouelette
February 17, 1984
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