Expanded genetic code

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https://en.wikipedia.org/wiki/Expanded_genetic_code 

 

There must not be crosstalk between the new tRNA/synthase pair and the existing tRNA/synthase molecules, only with the ribosomes

An expanded genetic code is an artificially modified genetic code in which one or more specific codons have been re-allocated to encode an amino acid that is not among the 22 common naturally-encoded proteinogenic amino acids.

The key prerequisites to expand the genetic code are:

• the non-standard amino acid to encode,
• an unused codon to adopt,
• a tRNA that recognises this codon, and
• a tRNA synthetase that recognises only that tRNA and only the non-standard amino acid.

Expanding the genetic code is an area of research of synthetic biology, an applied biological discipline whose goal is to engineer living systems for useful purposes. The genetic code expansion enriches the repertoire of useful tools available to science.

In May 2019, researchers, in a milestone effort, reported the creation of a new synthetic (possibly artificial) form of viable life, a variant of the bacteria Escherichia coli, by reducing the natural number of 64 codons in the bacterial genome to 61 codons (eliminating two out of the six codons coding for serine and one out of three stop codons) - of which 59 used to encode 20 amino acids.

Introduction

For vocabulary, see Glossary of gene expression terms. For a non-technical introduction to the topic, see Introduction to genetics.

It is noteworthy that the genetic code for all organisms is basically the same, so that all living beings use the same ’genetic language’. In general, the introduction of new functional unnatural amino acids into proteins of living cells breaks the universality of the genetic language, which ideally leads to alternative life forms. Proteins are produced thanks to the translational system molecules, which decode the RNA messages into a string of amino acids. The translation of genetic information contained in messenger RNA (mRNA) into a protein is catalysed by ribosomes. Transfer RNAs (tRNA) are used as keys to decode the mRNA into its encoded polypeptide. The tRNA recognizes a specific three nucleotide codon in the mRNA with a complementary sequence called the anticodon on one of its loops. Each three-nucleotide codon is translated into one of twenty naturally occurring amino acids. There is at least one tRNA for any codon, and sometimes multiple codons code for the same amino acid. Many tRNAs are compatible with several codons. An enzyme called an aminoacyl tRNA synthetase covalently attaches the amino acid to the appropriate tRNA. Most cells have a different synthetase for each amino acid (20 or more synthetases). On the other hand, some bacteria have fewer than 20 aminoacyl tRNA synthetases, and introduce the "missing" amino acid(s) by modification of a structurally related amino acid by an aminotransferase enzyme. A feature exploited in the expansion of the genetic code is the fact that the aminoacyl tRNA synthetase often does not recognize the anticodon, but another part of the tRNA, meaning that if the anticodon were to be mutated the encoding of that amino acid would change to a new codon. In the ribosome, the information in mRNA is translated into a specific amino acid when the mRNA codon matches with the complementary anticodon of a tRNA, and the attached amino acid is added onto a growing polypeptide chain. When it is released from the ribosome, the polypeptide chain folds into a functioning protein.

In order to incorporate a novel amino acid into the genetic code several changes are required. First, for successful translation of a novel amino acid, the codon to which the novel amino acid is assigned cannot already code for one of the 20 natural amino acids. Usually a nonsense codon (stop codon) or a four-base codon are used. Second, a novel pair of tRNA and aminoacyl tRNA synthetase are required, these are called the orthogonal set. The orthogonal set must not crosstalk with the endogenous tRNA and synthetase sets, while still being functionally compatible with the ribosome and other components of the translation apparatus. The active site of the synthetase is modified to accept only the novel amino acid. Most often, a library of mutant synthetases is screened for one which charges the tRNA with the desired amino acid. The synthetase is also modified to recognize only the orthogonal tRNA. The tRNA synthetase pair is often engineered in other bacteria or eukaryotic cells.

In this area of research, the 20 encoded proteinogenic amino acids are referred to as standard amino acids, or alternatively as natural or canonical amino acids, while the added amino acids are called non-standard amino acids (NSAAs), or unnatural amino acids (uAAs; term not used in papers dealing with natural non-proteinogenic amino acids, such as phosphoserine), or non-canonical amino acids.

Non-standard amino acids

Tyrosine and some synthetic tyrosine variants used for protein labeling. Different variants of tyrosine have been synthesized and can be incorporated into proteins using an expanded genetic code. The variants depicted here are all used for chemical or photochemical linking. This means that the incorporated AA specifically reacts with either a particular chemical group (such as hydrazides, amines, azides, or thiols) or can be UV-activated to crosslink with other AAs.

The first element of the system is the amino acid that is added to the genetic code of a certain strain of organism.

Over 71 different NSAAs have been added to different strains of E. coli, yeast or mammalian cells. Due to technical details (easier chemical synthesis of NSAAs, less crosstalk and easier evolution of the aminoacyl-tRNA synthase), the NSAAs are generally larger than standard amino acids and most often have a phenylalanine core but with a large variety of different substituents. These allow a large repertoire of new functions, such as labeling (see figure), as a fluorescent reporter (e.g. dansylalanine) or to produce translational proteins in E. coli with Eukaryotic post-translational modifications (e.g. phosphoserine, phosphothreonine, and phosphotyrosine).

The founding work was reported by Rolf Furtner, who singlehandedly used yeast tRNA^Phe/PheRS pair to incorporate p-iodophenylalanine in E.coli.

Unnatural amino acids incorporated into proteins include heavy atom-containing amino acids to facilitate certain x-ray crystallographic studies; amino acids with novel steric/packing and electronic properties; photocrosslinking amino acids which can be used to probe protein-protein interactions in vitro or in vivo; keto, acetylene, azide, and boronate-containing amino acids which can be used to selectively introduce a large number of biophysical probes, tags, and novel chemical functional groups into proteins in vitro or in vivo; redox active amino acids to probe and modulate electron transfer; photocaged and photoisomerizable amino acids to photoregulate biological processes; metal binding amino acids for catalysis and metal ion sensing; amino acids that contain fluorescent or infra-red active side chains to probe protein structure and dynamics; α-hydroxy acids and D-amino acids as probes of backbone conformation and hydrogen bonding interactions; and sulfated amino acids and mimetics of phosphorylated amino acids as probes of post-translational modifications.

Availability of the non-standard amino acid requires that the organism either import it from the medium or biosynthesize it. In the first case, the unnatural amino acid is first synthesized chemically in its optically pure L-form. It is then added to the growth medium of the cell. A library of compounds is usually tested for use in incorporation of the new amino acid, but this is not always necessary, for example, various transport systems can handle unnatural amino acids with apolar side-chains. In the second case, a biosynthetic pathway needs to be engineered, for example, an E. coli strain that biosynthesizes a novel amino acid (p-aminophenylalanine) from basic carbon sources and includes it in its genetic code. Another example: the production of phosphoserine, a natural metabolite, and consequently required alteration of its pathway flux to increase its production.

Codon assignment

Another element of the system is a codon to allocate to the new amino acid.

A major problem for the genetic code expansion is that there are no free codons. The genetic code has a non-random layout that shows tell-tale signs of various phases of primordial evolution, however, it has since frozen into place and is near-universally conserved. Nevertheless, some codons are rarer than others. In fact, in E. coli (and all organisms) the codon usage is not equal, but presents several rare codons (see table), the rarest being the amber stop codon (UAG).

Amber codon suppression

The possibility of reassigning codons was realized by Normanly et al. in 1990, when a viable mutant strain of E. coli read through the UAG ("amber") stop codon. This was possible thanks to the rarity of this codon and the fact that release factor 1 alone makes the amber codon terminate translation. Later, in the Schultz lab, the tRNATyr/tyrosyl-tRNA synthetase (TyrRS) from Methanococcus jannaschii, an archaebacterium, was used to introduce a tyrosine instead of STOP, the default value of the amber codon. This was possible because of the differences between the endogenous bacterial synthases and the orthologous archaeal synthase, which do not recognize each other. Subsequently, the group evolved the orthologonal tRNA/synthase pair to utilise the non-standard amino acid O-methyltyrosine. This was followed by the larger naphthylalanine and the photocrosslinking benzoylphenylalanine, which proved the potential utility of the system.

The amber codon is the least used codon in Escherichia coli, but hijacking it results in a substantial loss of fitness. One study, in fact, found that there were at least 83 peptides majorly affected by the readthrough Additionally, the labelling was incomplete. As a consequence, several strains have been made to reduce the fitness cost, including the removal of all amber codons from the genome. In most E. coli K-12 strains (viz. Escherichia coli (molecular biology) for strain pedigrees) there are 314 UAG stop codons. Consequently, a gargantuan amount of work has gone into the replacement of these. One approach pioneered by the group of Prof. George Church from Harvard, was dubbed MAGE in CAGE: this relied on a multiplex transformation and subsequent strain recombination to remove all UAG codons—the latter part presented a halting point in a first paper, but was overcome. This resulted in the E. coli strain C321.ΔA, which lacks all UAG codons and RF1. This allowed an experiment to be done with this strain to make it "addicted" to the amino acid biphenylalanine by evolving several key enzymes to require it structurally, therefore putting its expanded genetic code under positive selection.

Rare sense codon reassignment

In addition to the amber codon, rare sense codons have also been considered for use. The AGG codon codes for arginine, but a strain has been successfully modified to make it code for 6-N-allyloxycarbonyl-lysine. Another candidate is the AUA codon, which is unusual in that its respective tRNA has to differentiate against AUG that codes for methionine (primordially, isoleucine, hence its location). In order to do this, the AUA tRNA has a special base, lysidine. The deletion of the synthase (tilS) was possible thanks to the replacement of the native tRNA with that of Mycoplasma mobile (no lysidine). The reduced fitness is a first step towards pressuring the strain to lose all instances of AUA, allowing it to be used for genetic code expansion.

Four base (quadruplet) codons

While triplet codons are the basis of the genetic code in nature, programmed +1 frameshift is a natural process that allows the use of a four-nucleotide sequence (quadruplet codon) to encode an amino acid. Recent developments in genetic code engineering also showed that quadruplet codon could be used to encode non-standard amino acids under experimental conditions. This allowed the simultaneous usage of two unnatural amino acids, p-azidophenylalanine (pAzF) and N6-[(2-propynyloxy)carbonyl]lysine (CAK), which cross-link with each other by Huisgen cycloaddition. Quadrupled decoding in wild-type, non-recoded strains is very inefficient. This stems from the fact that the interaction between engineered tRNAs with ternary complexes or other translation components is not as favorable and strong as with cell endogenous translation elements. This problem can be overcome by specifically engineering and evolving tRNA that can decode quadruplet codons in non-recoded strains. Up to 4 different quadruplet orthogonal tRNA/tRNA synthethase pairs can be generated in this manner. Quadruplet codon decoding approach has also been applied to the construction of an HIV-1 vaccine.

tRNA/synthetase pair

Another key element is the tRNA/synthetase pair.

The orthologous set of synthetase and tRNA can be mutated and screened through directed evolution to charge the tRNA with a different, even novel, amino acid. Mutations to the plasmid containing the pair can be introduced by error-prone PCR or through degenerate primers for the synthetase's active site. Selection involves multiple rounds of a two-step process, where the plasmid is transferred into cells expressing chloramphenicol acetyl transferase with a premature amber codon. In the presence of toxic chloramphenicol and the non-natural amino acid, the surviving cells will have overridden the amber codon using the orthogonal tRNA aminoacylated with either the standard amino acids or the non-natural one. To remove the former, the plasmid is inserted into cells with a barnase gene (toxic) with a premature amber codon but without the non-natural amino acid, removing all the orthogonal synthases that do not specifically recognize the non-natural amino acid. In addition to the recoding of the tRNA to a different codon, they can be mutated to recognize a four-base codon, allowing additional free coding options. The non-natural amino acid, as a result, introduces diverse physicochemical and biological properties in order to be used as a tool to explore protein structure and function or to create novel or enhanced protein for practical purposes.

Several methods for selecting the synthetase that accepts only the non-natural amino acid have been developed. One of which is by using a combination of positive and negative selection

Orthogonal sets in model organisms

The orthogonal pairs of synthetase and tRNA that work for one organism may not work for another, as the synthetase may mis-aminoacylate endogenous tRNAs or the tRNA be mis-aminoacylated itself by an endogenous synthetase. As a result, the sets created to date differ between organisms.

Pair	Source	E. coli	Yeast	Mammals	Notes
tRNATyr-TyrRS	Methanococcus jannaschii	Yes	No	No
tRNALys–LysRS	Pyrococcus horikoshii	Yes	No	No
tRNAGlu–GluRS	Pyrococcus horikoshii	Yes	No	No
tRNALeu–LeuRS	tRNA: mutant Halobacterium sp. RS: Methanobacterium thermoautotrophicum	Yes	No	No
tRNAAmber-PylRS	Methanosarcina barkeri and Methanosarcina mazei	Yes	Yes	Yes
tRNAAmber-3-iodotyrosyl-RS	RS: variant Methanocaldococcus jannaschii aaRS	Yes	No	No
tRNATyr/Amber-TyrRS	Escherichia coli	No	Yes	No	Reported in 2003, mentioned in 2014 LeuRS
tRNAiMet-GlnRS	tRNA: human RS: Escherichia coli	No	Yes	No	Switched to Amber codon.
tRNAifMet-TyrRS	tRNA: Escherichia coli RS: S. cerevisiae	Yes	Yes	No	Switched to Amber codon.
tRNALeu/Amber-LeuRS	Escherichia coli	No	Yes	Yes	Reported in 2004 and mutated for 2-Aminooctanoic acid, o-methyl tyrosine, and o-nitrobenzyl cysteine. Evolved in yeast for 4,5-dimethoxy- 2-nitrobenzyl serine, tested in mice and mammalian cells with photo- sensitive 4,5-dimethoxy-2- nitrobenzyl-cysteine.
tRNATyr-TyrRS	Bacillus stearothermophilus	No	No	Yes
tRNATrp-TrpRS	Bacillus subtilis, RS modified	No	No	Yes	New AA is 5-OH Trp.

In 2017 a mouse engineered with an extended genetic code that can produce proteins with unnatural amino acids was reported.

Orthogonal ribosomes

Similarly to orthogonal tRNAs and aminoacyl tRNA synthetases (aaRSs), orthogonal ribosomes have been engineered to work in parallel to the natural ribosomes. Orthogonal ribosomes ideally use different mRNA transcripts than their natural counterparts and ultimately should draw on a separate pool of tRNA as well. This should alleviate some of the loss of fitness which currently still arises from techniques such as Amber codon suppression. Additionally, orthogonal ribosomes can be mutated and optimized for particular tasks, like the recognition of quadruplet codons. Such an optimization is not possible, or highly disadvantageous for natural ribosomes.

o-Ribosome

In 2005 three sets of ribosomes were published, which did not recognize natural mRNA, but instead translated a separate pool of orthogonal mRNA (o-mRNA). This was achieved by changing the recognition sequence of the mRNA, the Shine-Dalgarno sequence, and the corresponding recognition sequence in the 16S rRNA of ribosomes, the so-called Anti-Shine-Darlgarno-Sequence. This way the base pairing, which is usually lost if either sequence is mutated, stays available. However the mutations in the 16S rRNA were not limited to the obviously base-pairing nucleotides of the classical Anti-Shine-Darlgarno sequence.

Ribo-X

In 2007 the group of Jason W. Chin presented an orthogonal ribosome, which was optimized for Amber codon suppression. The 16S rRNA was mutated in such a way that it bound the release factor RF1 less strongly than the natural ribosome does. This ribosome did not eliminate the problem of lowered cell fitness caused by suppressed stop codons in natural proteins. However through the improved specificity it raised the yields of correctly synthesized target protein significantly (from ~20% to >60% percent for one amber codon to be suppressed and form <1% to >20% for two amber codons).

Ribo-Q

In 2010 the group of Jason W. Chin presented a further optimized version of the orthogonal ribosome. The Ribo-Q is a 16S rRNA optimized to recognize tRNAs, which have quadruplet anti-codons to recognize quadruplet codons, instead of the natural triplet codons. With this approach the number of possible codons rises from 64 to 256. Even accounting for a variety of stop codons, more than 200 different amino acids could potentially be encoded this way.

Ribosome stapling

The orthogonal ribosomes described above all focus on optimizing the 16S rRNA. Thus far, this optimized 16S rRNA was combined with natural large-subunits to form orthogonal ribosomes. If the 23S rRNA, the main RNA-component of the large ribosomal subunit, is to be optimized as well, it had to be assured, that there was no crosstalk in the assembly of orthogonal and natural ribosomes (see figureX B). To ensure that optimized 23S rRNA would only form into ribosomes with the optimized 16S rRNA, the two rRNAs were combined into one transcript. By inserting the sequence for the 23S rRNA into a loop-region of the 16S rRNA sequence, both subunits still adopt functioning folds. Since the two rRNAs are linked and thus in constant proximity, they preferably bind each other, not other free floating ribosomal subunits.

Engineered peptidyl transferase center

In 2014 it was shown that by altering the peptidyl transferase center of the 23S rRNA, ribosomes could be created which draw on orthogonal pools of tRNA. The 3’ end of tRNAs is universally conserved to be CCA. The two cytidines base pair with two guanines the 23S rRNA to bind the tRNA to the ribosome. This interaction is required for translational fidelity. However, by co-mutating the binding nucleotides in such a way, that they can still base pair, the translational fidelity can be conserved. The 3’-end of the tRNA is mutated from CCA to CGA, while two cytidine nucleotides in the ribosomes A- and P-sites are mutated to guanidine. This leads to ribosomes which do not accept naturally occurring tRNAs as substrates and to tRNAs, which cannot be used as substrate by natural ribosomes.
To use such tRNAs effectively, they would have to be aminoacylated by specific, orthogonal aaRSs. Most naturally occurring aaRSs recognize the 3’-end of their corresponding tRNA. aaRSs for these 3’-mutated tRNAs are not available yet. Thus far, this system has only been shown to work in an in-vitro translation setting where the aminoacylation of the orthogonal tRNA was achieved using so called “flexizymes”. Flexizymes are ribozymes with tRNA-amino-aclylation activity.

Applications

With an expanded genetic code, the unnatural amino acid can be genetically directed to any chosen site in the protein of interest. The high efficiency and fidelity of this process allows a better control of the placement of the modification compared to modifying the protein post-translationally, which, in general, will target all amino acids of the same type, such as the thiol group of cysteine and the amino group of lysine. Also, an expanded genetic code allows modifications to be carried out in vivo. The ability to site-specifically direct lab-synthesized chemical moieties into proteins allows many types of studies that would otherwise be extremely difficult, such as:

• Probing protein structure and function: By using amino acids with slightly different size such as O-methyltyrosine or dansylalanine instead of tyrosine, and by inserting genetically coded reporter moieties (color-changing and/or spin-active) into selected protein sites, chemical information about the protein's structure and function can be measured.
• Probing the role of post-translational modifications in protein structure and function: By using amino acids that mimic post-translational modifications such as phosphoserine, biologically active protein can be obtained, and the site-specific nature of the amino acid incorporation can lead to information on how the position, density, and distribution of protein phosphorylation effect protein function.
• Identifying and regulating protein activity: By using photocaged aminoacids, protein function can be "switched" on or off by illuminating the organism.
• Changing the mode of action of a protein: One can start with the gene for a protein that binds a certain sequence of DNA and, by inserting a chemically active amino acid into the binding site, convert it to a protein that cuts the DNA rather than binding it.
• Improving immunogenicity and overcoming self-tolerance: By replacing strategically chosen tyrosines with p-nitro phenylalanine, a tolerated self-protein can be made immunogenic.
• Selective destruction of selected cellular components: using an expanded genetic code, unnatural, destructive chemical moieties (sometimes called "chemical warheads") can be incorporated into proteins that target specific cellular components.
• Producing better protein: the evolution of T7 bacteriophages on a non-evolving E. coli strain that encoded 3-iodotyrosine on the amber codon, resulted in a population fitter than wild-type thanks to the presence of iodotyrosine in its proteome
• Probing protein localization and protein-protein interaction in bacteria.

Future

The expansion of the genetic code is still in its infancy. Current methodology uses only one non-standard amino acid at the time, whereas ideally multiple could be used. In fact, the group of Jason Chin has recently broken the record for a genetically recoded E.coli strain that can simultaneously incorporate up to 4 unnatural amino acids. Moreover, there has been development in software that allows combination of orthogonal ribosomes and unnatural tRNA/RS pairs in order to improve protein yield and fidelity.

Recoded synthetic genome

One way to achieve the encoding of multiple unnatural amino acids is by synthesising a rewritten genome.^[74] In 2010, at the cost of $40 million an organism, Mycoplasma laboratorium, was constructed that was controlled by a synthetic, but not recoded, genome. The first genetically recoded organism was created by a collaboration between George Church's and Farren Isaacs' labs, when the wild type E.coli MG1655 was recoded in such a way that all 321 known stop codons (UAG) were substituted with synonymous UAA codons and release factor 1 was knocked out in order to eliminate the interaction with the exogenous stop codon and improve unnatural protein synthesis. In 2019, Escherichia coli Syn61 was created, with a 4 megabase recoded genome consisting of only 61 codons instead of the natural 64. In addition to the elimination of the usage of rare codons, the specificity of the system needs to be increased as many tRNA recognise several codons.

Expanded genetic alphabet

Main article: Unnatural base pair

Another approach is to expand the number of nucleobases to increase the coding capacity.

An unnatural base pair (UBP) is a designed subunit (or nucleobase) of DNA which is created in a laboratory and does not occur in nature. A demonstration of UBPs were achieved in vitro by Ichiro Hirao's group at RIKEN institute in Japan. In 2002, they developed an unnatural base pair between 2-amino-8-(2-thienyl)purine (s) and pyridine-2-one (y) that functions in vitro in transcription and translation for the site-specific incorporation of non-standard amino acids into proteins. In 2006, they created 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) as a third base pair for replication and transcription. Afterward, Ds and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (Px) was discovered as a high fidelity pair in PCR amplification. In 2013, they applied the Ds-Px pair to DNA aptamer generation by in vitro selection (SELEX) and demonstrated the genetic alphabet expansion significantly augment DNA aptamer affinities to target proteins.

In 2012, a group of American scientists led by Floyd Romesberg, a chemical biologist at the Scripps Research Institute in San Diego, California, published that his team designed an unnatural base pair (UBP). The two new artificial nucleotides or Unnatural Base Pair (UBP) were named "d5SICS" and "dNaM." More technically, these artificial nucleotides bearing hydrophobic nucleobases, feature two fused aromatic rings that form a (d5SICS–dNaM) complex or base pair in DNA. In 2014 the same team from the Scripps Research Institute reported that they synthesized a stretch of circular DNA known as a plasmid containing natural T-A and C-G base pairs along with the best-performing UBP Romesberg's laboratory had designed, and inserted it into cells of the common bacterium E. coli that successfully replicated the unnatural base pairs through multiple generations. This is the first known example of a living organism passing along an expanded genetic code to subsequent generations. This was in part achieved by the addition of a supportive algal gene that expresses a nucleotide triphosphate transporter which efficiently imports the triphosphates of both d5SICSTP and dNaMTP into E. coli bacteria. Then, the natural bacterial replication pathways use them to accurately replicate the plasmid containing d5SICS–dNaM.

The successful incorporation of a third base pair into a living micro-organism is a significant breakthrough toward the goal of greatly expanding the number of amino acids which can be encoded by DNA, thereby expanding the potential for living organisms to produce novel proteins. The artificial strings of DNA do not encode for anything yet, but scientists speculate they could be designed to manufacture new proteins which could have industrial or pharmaceutical uses.

In May 2014, researchers announced that they had successfully introduced two new artificial nucleotides into bacterial DNA, and by including individual artificial nucleotides in the culture media, were able to passage the bacteria 24 times; they did not create mRNA or proteins able to use the artificial nucleotides.

Related methods

Selective pressure incorporation (SPI) method for production of alloproteins

There have been many studies that have produced protein with non-standard amino acids, but they do not alter the genetic code. These protein, called alloprotein, are made by incubating cells with an unnatural amino acid in the absence of a similar coded amino acid in order for the former to be incorporated into protein in place of the latter, for example L-2-aminohexanoic acid (Ahx) for methionine (Met).

These studies rely on the natural promiscuous activity of the aminoacyl tRNA synthetase to add to its target tRNA an unnatural amino acid (i.e. analog) similar to the natural substrate, for example methionyl-tRNA synthase's mistaking isoleucine for methionine. In protein crystallography, for example, the addition of selenomethionine to the media of a culture of a methionine-auxotrophic strain results in proteins containing selenomethionine as opposed to methionine (viz. Multi-wavelength anomalous dispersion for reason). Another example is that photoleucine and photomethionine are added instead of leucine and methionine to cross-label protein. Similarly, some tellurium-tolerant fungi can incorporate tellurocysteine and telluromethionine into their protein instead of cysteine and methionine. The objective of expanding the genetic code is more radical as it does not replace an amino acid, but it adds one or more to the code. On the other hand, proteome-wide replacements are most efficiently performed by global amino acid substitutions. For example, global proteome-wide substitutions of natural amino acids with fluorinated analogs have been attempted in E. coli and B. subtilis. A complete tryptophan substitution with thienopyrrole-alanine in response to 20899 UGG codons in E. coli was reported in 2015 by Budisa and Söll. Moreover, many biological phenomena, such as protein folding and stability, are based on synergistic effects at many positions in the protein sequence.

In this context, the SPI method generates recombinant protein variants or alloproteins directly by substitution of natural amino acids with unnatural counterparts. An amino acid auxotrophic expression host is supplemented with an amino acid analog during target protein expression. This approach avoids the pitfalls of suppression-based methods and it is superior to it in terms of efficiency, reproducibility and an extremely simple experimental setup. Numerous studies demonstrated how global substitution of canonical amino acids with various isosteric analogs caused minimal structural perturbations but dramatic changes in thermodynamic, folding, aggregation spectral properties and enzymatic activity.

in vitro synthesis

Main article: mRNA display

The genetic code expansion described above is in vivo. An alternative is the change of coding in vitro translation experiments. This requires the depletion of all tRNAs and the selective reintroduction of certain aminoacylated-tRNAs, some chemically aminoacylated.

Chemical synthesis

Main article: Peptide synthesis

There are several techniques to produce peptides chemically, generally it is by solid-phase protection chemistry. This means that any (protected) amino acid can be added into the nascent sequence.

In November 2017, a team from the Scripps Research Institute reported having constructed a semi-synthetic E. coli bacteria genome using six different nucleic acids (versus four found in nature). The two extra 'letters' form a third, unnatural base pair. The resulting organisms were able to thrive and synthesize proteins using "unnatural amino acids". The unnatural base pair used is dNaM–dTPT3. This unnatural base pair has been demonstrated previously, but this is the first report of transcription and translation of proteins using an unnatural base pair.

Cathodic protection

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Cathodic_protection 

 

Aluminium sacrificial anodes (light colored rectangular bars) mounted on a steel jacket structure.

 

Zinc sacrificial anode (rounded object) screwed to the underside of the hull of a small boat.

Cathodic protection is a technique used to control the corrosion of a metal surface by making it the cathode of an electrochemical cell. A simple method of protection connects the metal to be protected to a more easily corroded "sacrificial metal" to act as the anode. The sacrificial metal then corrodes instead of the protected metal. For structures such as long pipelines, where passive galvanic cathodic protection is not adequate, an external DC electrical power source is used to provide sufficient current.

Cathodic protection systems protect a wide range of metallic structures in various environments. Common applications are: steel water or fuel pipelines and steel storage tanks such as home water heaters; steel pier piles; ship and boat hulls; offshore oil platforms and onshore oil well casings; offshore wind farm foundations and metal reinforcement bars in concrete buildings and structures. Another common application is in galvanized steel, in which a sacrificial coating of zinc on steel parts protects them from rust.

Cathodic protection can, in some cases, prevent stress corrosion cracking.

History

Cathodic protection was first described by Sir Humphry Davy in a series of papers presented to the Royal Society in London in 1824. The first application was to HMS Samarang in 1824. Sacrificial anodes made from iron attached to the copper sheath of the hull below the waterline dramatically reduced the corrosion rate of the copper. However, a side effect of cathodic protection was the increase in marine growth. Usually, copper when corroding releases copper ions which have an anti-fouling effect. Since excess marine growth affected the performance of the ship, the Royal Navy decided that it was better to allow the copper to corrode and have the benefit of reduced marine growth, so cathodic protection was not used further.

Davy was assisted in his experiments by his pupil Michael Faraday, who continued his research after Davy's death. In 1834, Faraday discovered the quantitative connection between corrosion weight loss and electric current and thus laid the foundation for the future application of cathodic protection.

Thomas Edison experimented with impressed current cathodic protection on ships in 1890, but was unsuccessful due to the lack of a suitable current source and anode materials. It would be 100 years after Davy's experiment before cathodic protection was used widely on oil pipelines in the United States — cathodic protection was applied to steel gas pipelines beginning in 1928 and more widely in the 1930s.

Types

Galvanic sacrificial anode attached to the hull of a ship, showing corrosion.

Galvanic

In the application of passive cathodic protection, a galvanic anode, a piece of a more electrochemically "active" metal (more negative electrode potential), is attached to the vulnerable metal surface where it is exposed to an electrolyte. Galvanic anodes are selected because they have a more "active" voltage than the metal of the target structure (typically steel).

Concrete has a pH around 13. In this environment the steel reinforcement has a passive protective layer and remains largely stable. Galvanic systems are "constant potential" systems that aim to restore the concrete's natural protective environment by providing a high initial current to restore passivity. It then reverts to a lower sacrificial current while harmful negative Chloride ions migrate away from the steel and towards the positive anode. The anodes remain reactive through their lifetime (10-20 years typically) increasing current when the resistivity decreases due to corrosion hazards such as rainfall, temperature increases or flooding. The reactive nature of these anodes makes them an efficient choice.

Unlike ICCP systems steel constant polarization is not the goal, rather the restoration of the environment. Polarization of the target structure is caused by the electron flow from the anode to the cathode, so the two metals must have a good electrically conductive contact. The driving force for the cathodic protection current is the difference in electrode potential between the anode and the cathode. During the initial phase of high current, the potential of the steel surface is polarized (pushed) more negative protecting the steel which hydroxide ion generation at the steel surface and ionic migration restore the concrete environment.

Over time the galvanic anode continues to corrode, consuming the anode material until eventually it must be replaced.

Galvanic or sacrificial anodes are made in various shapes and sizes using alloys of zinc, magnesium and aluminium. ASTM International publishes standards on the composition and manufacturing of galvanic anodes.

In order for galvanic cathodic protection to work, the anode must possess a lower (that is, more negative) electrode potential than that of the cathode (the target structure to be protected). The table below shows a simplified galvanic series which is used to select the anode metal. The anode must be chosen from a material that is lower on the list than the material to be protected.

Metal	Potential with respect to a Cu:CuSO4 reference electrode in neutral pH environment (volts)
Carbon, Graphite, Coke	+0.3
Platinum	0 to −0.1
Mill scale on Steel	−0.2
High Silicon Cast Iron	−0.2
Copper, brass, bronze	−0.2
Mild steel in concrete	−0.2
Lead	−0.5
Cast iron (not graphitized)	−0.5
Mild steel (rusted)	−0.2 to −0.5
Mild steel (clean)	−0.5 to −0.8
Commercially pure aluminium	−0.8
Aluminium alloy (5% zinc)	−1.05
Zinc	−1.1
Magnesium Alloy (6% Al, 3% Zn, 0.15% Mn)	−1.6
Commercially Pure Magnesium	−1.75

Impressed current systems

Simple impressed current cathodic protection system. A source of DC electric current is used to help drive the protective electrochemical reaction.

In some cases, impressed current cathodic protection (ICCP) systems are used. These consist of anodes connected to a DC power source, often a transformer-rectifier connected to AC power. In the absence of an AC supply, alternative power sources may be used, such as solar panels, wind power or gas powered thermoelectric generators.

Anodes for ICCP systems are available in a variety of shapes and sizes. Common anodes are tubular and solid rod shapes or continuous ribbons of various materials. These include high silicon cast iron, graphite, mixed metal oxide (MMO), platinum and niobium coated wire and other materials.

For pipelines, anodes are arranged in groundbeds either distributed or in a deep vertical hole depending on several design and field condition factors including current distribution requirements.

Cathodic protection transformer-rectifier units are often custom manufactured and equipped with a variety of features, including remote monitoring and control, integral current interrupters and various type of electrical enclosures. The output DC negative terminal is connected to the structure to be protected by the cathodic protection system. The rectifier output DC positive cable is connected to the anodes. The AC power cable is connected to the rectifier input terminals.

The output of the ICCP system should be optimised to provide enough current to provide protection to the target structure. Some cathodic protection transformer-rectifier units are designed with taps on the transformer windings and jumper terminals to select the voltage output of the ICCP system. Cathodic protection transformer-rectifier units for water tanks and used in other applications are made with solid state circuits to automatically adjust the operating voltage to maintain the optimum current output or structure-to-electrolyte potential. Analog or digital meters are often installed to show the operating voltage (DC and sometime AC) and current output. For shore structures and other large complex target structures, ICCP systems are often designed with multiple independent zones of anodes with separate cathodic protection transformer-rectifier circuits.

Hybrid Systems

Hybrid systems have been used for over a decade and incorporate the coordination, monitoring and high restorative current flow of ICCP systems with the reactive, lower cost and easier to maintain galvanic anodes.

The system is made up of wired galvanic anodes in arrays typically 400mm apart which are then initially powered for a short period to restore the concrete and power ionic migration. The power supply is then taken away and the anodes simply attached to the steel as a galvanic system. More powered phases can be administered if needed. Like Galvanic systems corrosion rate monitoring from polarisation tests and half-cell potential mapping can be used to measure corrosion. Polarization is not the goal for the life of the system.

Applications

Hot Water Tank / Water Heater

This technology is also used to protect water heaters. Indeed, the electrons sent by the imposed current anode (composed of titanium and covered with MMO) prevents the inside of the tank from rusting.

In order to be recognized as effective, these anodes must comply with certain standards: A cathodic protection system is considered efficient when its potential reaches or exceeds the limits established by the cathodic protection criteria. The cathode protection criteria used come from the standard NACE SP0388-2007 (formerly RP0388-2001) of the NACE National Association of Corrosion Engineers.

Pipelines

An air cooled cathodic protection rectifier connected to a pipeline.

 

Cathodic protection markers over a gas pipeline in Leeds, West Yorkshire, England.

Hazardous product pipelines are routinely protected by a coating supplemented with cathodic protection. An impressed current cathodic protection system (ICCP) for a pipeline consists of a DC power source, often an AC powered transformer rectifier and an anode, or array of anodes buried in the ground (the anode groundbed).

The DC power source would typically have a DC output of up to 50 amperes and 50 volts, but this depends on several factors, such as the size of the pipeline and coating quality. The positive DC output terminal would be connected via cables to the anode array, while another cable would connect the negative terminal of the rectifier to the pipeline, preferably through junction boxes to allow measurements to be taken.

Anodes can be installed in a groundbed consisting of a vertical hole backfilled with conductive coke (a material that improves the performance and life of the anodes) or laid in a prepared trench, surrounded by conductive coke and backfilled. The choice of groundbed type and size depends on the application, location and soil resistivity.

The DC cathodic protection current is then adjusted to the optimum level after conducting various tests including measurements of pipe-to-soil potentials or electrode potential.

It is sometimes more economically viable to protect a pipeline using galvanic (sacrificial) anodes. This is often the case on smaller diameter pipelines of limited length. Galvanic anodes rely on the galvanic series potentials of the metals to drive cathodic protection current from the anode to the structure being protected.

Water pipelines of various pipe materials are also provided with cathodic protection where owners determine the cost is reasonable for the expected pipeline service life extension attributed to the application of cathodic protection.

Ships and boats

The white patches visible on the ship's hull are zinc block sacrificial anodes.

Cathodic protection on ships is often implemented by galvanic anodes attached to the hull and ICCP for larger vessels. Since ships are regularly removed from the water for inspections and maintenance, it is a simple task to replace the galvanic anodes.

Galvanic anodes are generally shaped to reduced drag in the water and fitted flush to the hull to also try to minimize drag.

Smaller vessels, with non-metallic hulls, such as yachts, are equipped with galvanic anodes to protect areas such as outboard motors. As with all galvanic cathodic protection, this application relies on a solid electrical connection between the anode and the item to be protected.

For ICCP on ships, the anodes are usually constructed of a relatively inert material such as platinised titanium. A DC power supply is provided within the ship and the anodes mounted on the outside of the hull. The anode cables are introduced into the ship via a compression seal fitting and routed to the DC power source. The negative cable from the power supply is simply attached to the hull to complete the circuit. Ship ICCP anodes are flush-mounted, minimizing the effects of drag on the ship, and located a minimum 5 ft below the light load line in an area to avoid mechanical damage. The current density required for protection is a function of velocity and considered when selecting the current capacity and location of anode placement on the hull.

Some ships may require specialist treatment, for example aluminium hulls with steel fixtures will create an electrochemical cell where the aluminium hull can act as a galvanic anode and corrosion is enhanced. In cases like this, aluminium or zinc galvanic anodes can be used to offset the potential difference between the aluminium hull and the steel fixture. If the steel fixtures are large, several galvanic anodes may be required, or even a small ICCP system.

Marine

Marine cathodic protection covers many areas, jetties, harbors, offshore structures. The variety of different types of structure leads to a variety of systems to provide protection. Galvanic anodes are favored, but ICCP can also often be used. Because of the wide variety of structure geometry, composition, and architecture, specialized firms are often required to engineer structure-specific cathodic protection systems. Sometimes marine structures require retroactive modification to be effectively protected 

Steel in concrete

The application to concrete reinforcement is slightly different in that the anodes and reference electrodes are usually embedded in the concrete at the time of construction when the concrete is being poured. The usual technique for concrete buildings, bridges and similar structures is to use ICCP, but there are systems available that use the principle of galvanic cathodic protection as well, although in the UK at least, the use of galvanic anodes for atmospherically exposed reinforced concrete structures is considered experimental.

For ICCP, the principle is the same as any other ICCP system. However, in a typical atmospherically exposed concrete structure such as a bridge, there will be many more anodes distributed through the structure as opposed to an array of anodes as used on a pipeline. This makes for a more complicated system and usually an automatically controlled DC power source is used, possibly with an option for remote monitoring and operation. For buried or submerged structures, the treatment is similar to that of any other buried or submerged structure.

Galvanic systems offer the advantage of being easier to retrofit and do not need any control systems as ICCP does.

For pipelines constructed from pre-stressed concrete cylinder pipe (PCCP), the techniques used for cathodic protection are generally as for steel pipelines except that the applied potential must be limited to prevent damage to the prestressing wire.

The steel wire in a PCCP pipeline is stressed to the point that any corrosion of the wire can result in failure. An additional problem is that any excessive hydrogen ions as a result of an excessively negative potential can cause hydrogen embrittlement of the wire, also resulting in failure. The failure of too many wires will result in catastrophic failure of the PCCP. To implement ICCP therefore requires very careful control to ensure satisfactory protection. A simpler option is to use galvanic anodes, which are self-limiting and need no control.

Internal cathodic protection

Vessels, pipelines and tanks which are used to store or transport liquids can also be protected from corrosion on their internal surfaces by the use of cathodic protection. ICCP and galvanic systems can be used. A common application of internal cathodic protection is water storage tanks and power plant shell and tube heat exchangers.

Galvanized steel

Galvanizing generally refers to hot-dip galvanizing which is a way of coating steel with a layer of metallic zinc or tin. Galvanized coatings are quite durable in most environments because they combine the barrier properties of a coating with some of the benefits of cathodic protection. If the zinc coating is scratched or otherwise locally damaged and steel is exposed, the surrounding areas of zinc coating form a galvanic cell with the exposed steel and protect it from corrosion. This is a form of localized cathodic protection - the zinc acts as a sacrificial anode.

Galvanizing, while using the electrochemical principle of cathodic protection, is not actually cathodic protection. Cathodic protection requires the anode to be separate from the metal surface to be protected, with an ionic connection through the electrolyte and an electron connection through a connecting cable, bolt or similar. This means that any area of the protected structure within the electrolyte can be protected, whereas in the case of galvanizing, only areas very close to the zinc are protected. Hence, a larger area of bare steel would only be protected around the edges.

Automobiles

Several companies market electronic devices claiming to mitigate corrosion for automobiles and trucks. Corrosion control professionals find they do not work. There is no peer reviewed scientific testing and validation supporting the use of the devices. In 1996 the FTC ordered David McCready, a person that sold devices claiming to protect cars from corrosion, to pay restitution and banned the names "Rust Buster" and "Rust Evader."

Testing

Electrode potential is measured with reference electrodes. Copper-copper sulphate electrodes are used for structures in contact with soil or fresh water. Silver/silver chloride/seawater electrodes or pure zinc electrodes are used for seawater applications. The methods are described in EN 13509:2003 and NACE TM0497 along with the sources of error in the voltage that appears on the display of the meter. Interpretation of electrode potential measurements to determine the potential at the interface between the anode of the corrosion cell and the electrolyte requires training and cannot be expected to match the accuracy of measurements done in laboratory work.

Problems

Production of hydrogen

A side effect of improperly applied cathodic protection is the production of atomic hydrogen, leading to its absorption in the protected metal and subsequent hydrogen embrittlement of welds and materials with high hardness. Under normal conditions, the atomic hydrogen will combine at the metal surface to create hydrogen gas, which cannot penetrate the metal. Hydrogen atoms, however, are small enough to pass through the crystalline steel structure, and lead in some cases to hydrogen embrittlement.

Cathodic disbonding

This is a process of disbondment of protective coatings from the protected structure (cathode) due to the formation of hydrogen ions over the surface of the protected material (cathode). Disbonding can be exacerbated by an increase in alkali ions and an increase in cathodic polarization. The degree of disbonding is also reliant on the type of coating, with some coatings affected more than others. Cathodic protection systems should be operated so that the structure does not become excessively polarized, since this also promotes disbonding due to excessively negative potentials. Cathodic disbonding occurs rapidly in pipelines that contain hot fluids because the process is accelerated by heat flow.

Cathodic shielding

Effectiveness of cathodic protection (CP) systems on steel pipelines can be impaired by the use of solid film backed dielectric coatings such as polyethylene tapes, shrinkable pipeline sleeves, and factory applied single or multiple solid film coatings. This phenomenon occurs because of the high electrical resistivity of these film backings. Protective electric current from the cathodic protection system is blocked or shielded from reaching the underlying metal by the highly resistive film backing. Cathodic shielding was first defined in the 1980s as being a problem, and technical papers on the subject have been regularly published since then.

A 1999 report concerning a 20,600 bbl (3,280 m³) spill from a Saskatchewan crude oil line contains an excellent definition of the cathodic shielding problem:

"The triple situation of disbondment of the (corrosion) coating, the dielectric nature of the coating and the unique electrochemical environment established under the exterior coating, which acts as a shield to the electrical CP current, is referred to as CP shielding. The combination of tenting and disbondment permits a corrosive environment around the outside of the pipe to enter into the void between the exterior coating and the pipe surface. With the development of this CP shielding phenomenon, impressed current from the CP system cannot access exposed metal under the exterior coating to protect the pipe surface from the consequences of an aggressive corrosive environment. The CP shielding phenomenon induces changes in the potential gradient of the CP system across the exterior coating, which are further pronounced in areas of insufficient or sub-standard CP current emanating from the pipeline's CP system. This produces an area on the pipeline of insufficient CP defense against metal loss aggravated by an exterior corrosive environment."

Cathodic shielding is referenced in a number of the standards listed below. Newly issued USDOT regulation Title 49 CFR 192.112, in the section for Additional design requirements for steel pipe using alternative maximum allowable operating pressure requires that "The pipe must be protected against external corrosion by a non-shielding coating" (see coatings section on standard). Also, the NACE SP0169:2007 standard defines shielding in section 2, cautions against the use of materials that create electrical shielding in section 4.2.3, cautions against use of external coatings that create electrical shielding in section 5.1.2.3, and instructs readers to take 'appropriate action' when the effects of electrical shielding of cathodic protection current are detected on an operating pipeline in section 10.9.

Standards

• 49 CFR 192.451 - Requirements for Corrosion Control - Transportation of natural and other gas by pipeline: US minimum federal safety standards
• 49 CFR 195.551 - Requirements for Corrosion Control - Transportation of hazardous liquids by pipelines: US minimum federal safety standards
• AS 2832 - Australian Standards for Cathodic Protection
• ASME B31Q 0001-0191
• ASTM G 8, G 42 - Evaluating Cathodic Disbondment resistance of coatings
• DNV-RP-B401 - Cathodic Protection Design - Det Norske Veritas
• EN 12068:1999 - Cathodic protection. External organic coatings for the corrosion protection of buried or immersed steel pipelines used in conjunction with cathodic protection. Tapes and shrinkable materials
• EN 12473:2000 - General principles of cathodic protection in sea water
• EN 12474:2001 - Cathodic protection for submarine pipelines
• EN 12495:2000 - Cathodic protection for fixed steel offshore structures
• EN 12499:2003 - Internal cathodic protection of metallic structures
• EN 12696:2012 - Cathodic protection of steel in concrete
• EN 12954:2001 - Cathodic protection of buried or immersed metallic structures. General principles and application for pipelines
• EN 13173:2001 - Cathodic protection for steel offshore floating structures
• EN 13174:2001 - Cathodic protection for "Harbour Installations".
• EN 13509:2003 - Cathodic protection measurement techniques
• EN 13636:2004 - Cathodic protection of buried metallic tanks and related piping
• EN 14505:2005 - Cathodic protection of complex structures
• EN 15112:2006 - External cathodic protection of well casing
• EN 15280-2013 - Evaluation of a.c. corrosion likelihood of buried pipelines
• EN 50162:2004 - Protection against corrosion by stray current from direct current systems
• BS 7361-1:1991 - Cathodic Protection
• NACE SP0169:2013 - Control of External Corrosion on Underground or Submerged Metallic Piping Systems
• NACE TM 0497 - Measurement Techniques Related to Criteria for Cathodic Protection on Underground or Submerged Metallic Piping Systems

Biological warfare

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Biological_warfare 

Weapons of mass destruction
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Biological warfare, also known as germ warfare, is the use of biological toxins or infectious agents such as bacteria, viruses, insects, and fungi with the intent to kill, harm or incapacitate humans, animals or plants as an act of war. Biological weapons (often termed "bio-weapons", "biological threat agents", or "bio-agents") are living organisms or replicating entities ( ⁠i.e. viruses, which are not universally considered "alive"). Entomological (insect) warfare is a subtype of biological warfare.

Offensive biological warfare is prohibited under customary international humanitarian law and several international treaties. In particular, the 1972 Biological Weapons Convention (BWC) bans the development, production, acquisition, transfer, stockpiling and use of biological weapons. Therefore, the use of biological agents in armed conflict is a war crime. In contrast, defensive biological research for prophylactic, protective or other peaceful purposes is not prohibited by the BWC.

Biological warfare is distinct from warfare involving other types of weapons of mass destruction (WMD), including nuclear warfare, chemical warfare, and radiological warfare. None of these are considered conventional weapons, which are deployed primarily for their explosive, kinetic, or incendiary potential.

Biological weapons may be employed in various ways to gain a strategic or tactical advantage over the enemy, either by threats or by actual deployments. Like some chemical weapons, biological weapons may also be useful as area denial weapons. These agents may be lethal or non-lethal, and may be targeted against a single individual, a group of people, or even an entire population. They may be developed, acquired, stockpiled or deployed by nation states or by non-national groups. In the latter case, or if a nation-state uses it clandestinely, it may also be considered bioterrorism.

Biological warfare and chemical warfare overlap to an extent, as the use of toxins produced by some living organisms is considered under the provisions of both the BWC and the Chemical Weapons Convention. Toxins and psychochemical weapons are often referred to as midspectrum agents. Unlike bioweapons, these midspectrum agents do not reproduce in their host and are typically characterized by shorter incubation periods.

Overview

A biological attack could conceivably result in large numbers of civilian casualties and cause severe disruption to economic and societal infrastructure.

A nation or group that can pose a credible threat of mass casualty has the ability to alter the terms under which other nations or groups interact with it. When indexed to weapon mass and cost of development and storage, biological weapons possess destructive potential and loss of life far in excess of nuclear, chemical or conventional weapons. Accordingly, biological agents are potentially useful as strategic deterrents, in addition to their utility as offensive weapons on the battlefield.

As a tactical weapon for military use, a significant problem with biological warfare is that it would take days to be effective, and therefore might not immediately stop an opposing force. Some biological agents (smallpox, pneumonic plague) have the capability of person-to-person transmission via aerosolized respiratory droplets. This feature can be undesirable, as the agent(s) may be transmitted by this mechanism to unintended populations, including neutral or even friendly forces. Worse still, such a weapon could "escape" the laboratory where it was developed, even if there was no intent to use it – for example by infecting a researcher who then transmits it to the outside world before realizing that they were infected. Several cases are known of researchers becoming infected and dying of Ebola, which they had been working with in the lab (though nobody else was infected in those cases) – while there is no evidence that their work was directed towards biological warfare, it demonstrates the potential for accidental infection even of careful researchers fully aware of the dangers. While containment of biological warfare is less of a concern for certain criminal or terrorist organizations, it remains a significant concern for the military and civilian populations of virtually all nations.

History

Main article: History of biological warfare

War

History

Battlespace

Weapons

Tactics

Operational

Strategy

Grand strategy

Administrative

Organization

Personnel

Logistics

Science

Law

Theory

Antiquity and Middle Ages

Rudimentary forms of biological warfare have been practiced since antiquity. The earliest documented incident of the intention to use biological weapons is recorded in Hittite texts of 1500–1200 BCE, in which victims of tularemia were driven into enemy lands, causing an epidemic. The Assyrians poisoned enemy wells with the fungus ergot, though with unknown results. Scythian archers dipped their arrows and Roman soldiers their swords into excrements and cadavers – victims were commonly infected by tetanus as result. In 1346, the bodies of Mongol warriors of the Golden Horde who had died of plague were thrown over the walls of the besieged Crimean city of Kaffa. Specialists disagree about whether this operation was responsible for the spread of the Black Death into Europe, Near East and North Africa, resulting in the deaths of approximately 25 million Europeans.

Biological agents were extensively used in many parts of Africa from the sixteenth century AD, most of the time in the form of poisoned arrows, or powder spread on the war front as well as poisoning of horses and water supply of the enemy forces. In Borgu, there were specific mixtures to kill, hypnotize, make the enemy bold, and to act as an antidote against the poison of the enemy as well. The creation of biologicals was reserved for a specific and professional class of medicine-men.

1763 to present

The British Army attempted use of smallpox against Native Americans during the Siege of Fort Pitt in June 1763. A reported outbreak that began the spring before left as many as one hundred Native Americans dead in Ohio Country from 1763 to 1764. It is not clear whether the smallpox was a result of the Fort Pitt incident or the virus was already present among the Delaware people as outbreaks happened on their own every dozen or so years and the delegates were met again later and seemingly had not contracted smallpox. During the American Revolutionary War General George Washington heard a report that British General William Howe was going to send people inoculated with smallpox out from Boston, in order to infect other Americans. Washington reported this to Congress but said he could hardly give credit to it. Washington inoculated his soldiers, diminishing the effect of the on-going smallpox epidemic. Some historians believe that the British Marines deliberately used smallpox in New South Wales, Australia, in 1789. Dr Seth Carus states: "Ultimately, we have a strong circumstantial case supporting the theory that someone deliberately introduced smallpox in the Aboriginal population."

By 1900 the germ theory and advances in bacteriology brought a new level of sophistication to the techniques for possible use of bio-agents in war. Biological sabotage in the form of anthrax and glanders was undertaken on behalf of the Imperial German government during World War I (1914–1918), with indifferent results. The Geneva Protocol of 1925 prohibited the use of chemical and biological weapons.

With the onset of World War II, the Ministry of Supply in the United Kingdom established a biological warfare program at Porton Down, headed by the microbiologist Paul Fildes. The research was championed by Winston Churchill and soon tularemia, anthrax, brucellosis, and botulism toxins had been effectively weaponized. In particular, Gruinard Island in Scotland, was contaminated with anthrax during a series of extensive tests for the next 56 years. Although the UK never offensively used the biological weapons it developed, its program was the first to successfully weaponize a variety of deadly pathogens and bring them into industrial production. Other nations, notably France and Japan, had begun their own biological weapons programs.

When the United States entered the war, Allied resources were pooled at the request of the British. The U.S. then established a large research program and industrial complex at Fort Detrick, Maryland in 1942 under the direction of George W. Merck. The biological and chemical weapons developed during that period were tested at the Dugway Proving Grounds in Utah. Soon there were facilities for the mass production of anthrax spores, brucellosis, and botulism toxins, although the war was over before these weapons could be of much operational use.

Shiro Ishii, commander of Unit 731, which performed live human vivisections and other biological experimentation

The most notorious program of the period was run by the secret Imperial Japanese Army Unit 731 during the war, based at Pingfan in Manchuria and commanded by Lieutenant General Shirō Ishii. This unit biological warfare, conducted often fatal human experiments on prisoners, and produced biological weapons for combat use. Although the Japanese effort lacked the technological sophistication of the American or British programs, it far outstripped them in its widespread application and indiscriminate brutality. Biological weapons were used against Chinese soldiers and civilians in several military campaigns. In 1940, the Japanese Army Air Force bombed Ningbo with ceramic bombs full of fleas carrying the bubonic plague. Many of these operations were ineffective due to inefficient delivery systems, although up to 400,000 people may have died. During the Zhejiang-Jiangxi Campaign in 1942, around 1,700 Japanese troops died out of a total 10,000 Japanese soldiers who fell ill with disease when their own biological weapons attack rebounded on their own forces.

During the final months of World War II, Japan planned to use plague as a biological weapon against U.S. civilians in San Diego, California, during Operation Cherry Blossoms at Night. The plan was set to launch on 22 September 1945, but it was not executed because of Japan's surrender on 15 August 1945.

In Britain, the 1950s saw the weaponization of plague, brucellosis, tularemia and later equine encephalomyelitis and vaccinia viruses, but the programme was unilaterally cancelled in 1956. The United States Army Biological Warfare Laboratories weaponized anthrax, tularemia, brucellosis, Q-fever and others.

In 1969, US President Richard Nixon decided to unilaterally terminate the offensive biological weapons program of the US, allowing only scientific research for defensive measures. This decision increased the momentum of the negotiations for a ban on biological warfare, which took place from 1969 to 1972 in the United Nation's Conference of the Committee on Disarmament in Geneva. These negotiations resulted in the Biological Weapons Convention, which was opened for signature on 10 April 1972 and entered into force on 26 March 1975 after the ratification by 22 states.

Despite being a party and depositary to the BWC, the Soviet Union continued and expanded its massive offensive biological weapons program, under the leadership of the allegedly civilian institution Biopreparat. The Soviet Union attracted international suspicion after the 1979 Sverdlovsk anthrax leak killed approximately 65 to 100 people.

International law

Main articles: Geneva Protocol and Biological Weapons Convention

 

The Biological Weapons Convention

International restrictions on biological warfare began with the 1925 Geneva Protocol, which prohibits the use but not the possession or development of biological and chemical weapons. Upon ratification of the Geneva Protocol, several countries made reservations regarding its applicability and use in retaliation. Due to these reservations, it was in practice a "no-first-use" agreement only.

The 1972 Biological Weapons Convention (BWC) supplements the Geneva Protocol by prohibiting the development, production, acquisition, transfer, stockpiling and use of biological weapons. Having entered into force on 26 March 1975, the BWC was the first multilateral disarmament treaty to ban the production of an entire category of weapons of mass destruction. As of March 2021, 183 states have become party to the treaty. The BWC is considered to have established a strong global norm against biological weapons, which is reflected in the treaty's preamble, stating that the use of biological weapons would be "repugnant to the conscience of mankind". The BWC's effectiveness has been limited due to insufficient institutional support and the absence of any formal verification regime to monitor compliance.

In 1985, the Australia Group was established, a multilateral export control regime of 43 countries aiming to prevent the proliferation of chemical and biological weapons.

In 2004, the United Nations Security Council passed Resolution 1540, which obligates all UN Member States to develop and enforce appropriate legal and regulatory measures against the proliferation of chemical, biological, radiological, and nuclear weapons and their means of delivery, in particular, to prevent the spread of weapons of mass destruction to non-state actors.

Bioterrorism

Main article: Bioterrorism

Biological weapons are difficult to detect, economical and easy to use, making them appealing to terrorists. The cost of a biological weapon is estimated to be about 0.05 percent the cost of a conventional weapon in order to produce similar numbers of mass casualties per kilometer square. Moreover, their production is very easy as common technology can be used to produce biological warfare, like that used in production of vaccines, foods, spray devices, beverages and antibiotics. A major factor in biological warfare that attracts terrorists is that they can easily escape before the government agencies or secret agencies have even started their investigation. This is because the potential organism has an incubation period of 3 to 7 days, after which the results begin to appear, thereby giving terrorists a lead.

A technique called Clustered, Regularly Interspaced, Short Palindromic Repeat (CRISPR-Cas9) is now so cheap and widely available that scientists fear that the amateurs will start experimenting with them. In this technique, a DNA sequence is cut off and replaced with a new sequence or code that codes for a particular protein or characteristic, which could potentially show up in the required organism. Though this technique is a breakthrough and is commendable, it can cause serious issues and potential danger if used by people with wrong intentions. Concerns have emerged regarding do-it-yourself biology research organizations due to their associated risk that a rogue amateur DIY researcher could attempt to develop dangerous bioweapons using genome editing technology.

In 2002, when CNN went through Al-Qaeda's (AQ's) experiments with crude poisons, they found out that AQ had begun planning ricin and cyanide attacks with the help of a loose association of terrorist cells. The associates had infiltrated many countries like Turkey, Italy, Spain, France and others. In 2015, to combat the threat of bioterrorism, a National Blueprint for Biodefense was issued by the Blue-Ribbon Study Panel on Biodefense. Also, 233 potential exposures of select biological agents outside of the primary barriers of the biocontainment in the US were described by the annual report of the Federal Select Agent Program.

Though a verification system can reduce bioterrorism, an employee, or a lone terrorist having adequate knowledge of the company facilities, can cause potential danger by injecting a deadly or harmful substance into the facility. Moreover, it has been found that about 95% of accidents that have occurred due to low security have been done by employees or those who had a security clearance.

Entomology

Main article: Entomological warfare

Entomological warfare (EW) is a type of biological warfare that uses insects to attack the enemy. The concept has existed for centuries and research and development have continued into the modern era. EW has been used in battle by Japan and several other nations have developed and been accused of using an entomological warfare program. EW may employ insects in a direct attack or as vectors to deliver a biological agent, such as plague. Essentially, EW exists in three varieties. One type of EW involves infecting insects with a pathogen and then dispersing the insects over target areas. The insects then act as a vector, infecting any person or animal they might bite. Another type of EW is a direct insect attack against crops; the insect may not be infected with any pathogen but instead represents a threat to agriculture. The final method uses uninfected insects, such as bees or wasps, to directly attack the enemy.

Genetics

Theoretically, novel approaches in biotechnology, such as synthetic biology could be used in the future to design novel types of biological warfare agents.

1. Would demonstrate how to render a vaccine ineffective;
2. Would confer resistance to therapeutically useful antibiotics or antiviral agents;
3. Would enhance the virulence of a pathogen or render a nonpathogen virulent;
4. Would increase the transmissibility of a pathogen;
5. Would alter the host range of a pathogen;
6. Would enable the evasion of diagnostic/detection tools;
7. Would enable the weaponization of a biological agent or toxin.

Most of the biosecurity concerns in synthetic biology are focused on the role of DNA synthesis and the risk of producing genetic material of lethal viruses (e.g. 1918 Spanish flu, polio) in the lab. Recently, the CRISPR/Cas system has emerged as a promising technique for gene editing. It was hailed by The Washington Post as "the most important innovation in the synthetic biology space in nearly 30 years." While other methods take months or years to edit gene sequences, CRISPR speeds that time up to weeks. Due to its ease of use and accessibility, it has raised a number of ethical concerns, especially surrounding its use in the biohacking space.

By target

Anti-personnel

The international biological hazard symbol

Ideal characteristics of a biological agent to be used as a weapon against humans are high infectivity, high virulence, non-availability of vaccines and availability of an effective and efficient delivery system. Stability of the weaponized agent (the ability of the agent to retain its infectivity and virulence after a prolonged period of storage) may also be desirable, particularly for military applications, and the ease of creating one is often considered. Control of the spread of the agent may be another desired characteristic.

The primary difficulty is not the production of the biological agent, as many biological agents used in weapons can be manufactured relatively quickly, cheaply and easily. Rather, it is the weaponization, storage, and delivery in an effective vehicle to a vulnerable target that pose significant problems.

For example, Bacillus anthracis is considered an effective agent for several reasons. First, it forms hardy spores, perfect for dispersal aerosols. Second, this organism is not considered transmissible from person to person, and thus rarely if ever causes secondary infections. A pulmonary anthrax infection starts with ordinary influenza-like symptoms and progresses to a lethal hemorrhagic mediastinitis within 3–7 days, with a fatality rate that is 90% or higher in untreated patients. Finally, friendly personnel and civilians can be protected with suitable antibiotics.

Agents considered for weaponization, or known to be weaponized, include bacteria such as Bacillus anthracis, Brucella spp., Burkholderia mallei, Burkholderia pseudomallei, Chlamydophila psittaci, Coxiella burnetii, Francisella tularensis, some of the Rickettsiaceae (especially Rickettsia prowazekii and Rickettsia rickettsii), Shigella spp., Vibrio cholerae, and Yersinia pestis. Many viral agents have been studied and/or weaponized, including some of the Bunyaviridae (especially Rift Valley fever virus), Ebolavirus, many of the Flaviviridae (especially Japanese encephalitis virus), Machupo virus, Coronaviruses (especially SARS-Cov-2 that causes COVID-19), Marburg virus, Variola virus, and yellow fever virus. Fungal agents that have been studied include Coccidioides spp.

Toxins that can be used as weapons include ricin, staphylococcal enterotoxin B, botulinum toxin, saxitoxin, and many mycotoxins. These toxins and the organisms that produce them are sometimes referred to as select agents. In the United States, their possession, use, and transfer are regulated by the Centers for Disease Control and Prevention's Select Agent Program.

The former US biological warfare program categorized its weaponized anti-personnel bio-agents as either Lethal Agents (Bacillus anthracis, Francisella tularensis, Botulinum toxin) or Incapacitating Agents (Brucella suis, Coxiella burnetii, Venezuelan equine encephalitis virus, Staphylococcal enterotoxin B).

Anti-agriculture

Anti-crop/anti-vegetation/anti-fisheries

See also: United States herbicidal warfare research

The United States developed an anti-crop capability during the Cold War that used plant diseases (bioherbicides, or mycoherbicides) for destroying enemy agriculture. Biological weapons also target fisheries as well as water-based vegetation. It was believed that the destruction of enemy agriculture on a strategic scale could thwart Sino-Soviet aggression in a general war. Diseases such as wheat blast and rice blast were weaponized in aerial spray tanks and cluster bombs for delivery to enemy watersheds in agricultural regions to initiate epiphytotic (epidemics among plants). On the other hand, some sources report that these agents were stockpiled but never weaponized. When the United States renounced its offensive biological warfare program in 1969 and 1970, the vast majority of its biological arsenal was composed of these plant diseases. Enterotoxins and Mycotoxins were not affected by Nixon's order.

Though herbicides are chemicals, they are often grouped with biological warfare and chemical warfare because they may work in a similar manner as biotoxins or bioregulators. The Army Biological Laboratory tested each agent and the Army's Technical Escort Unit was responsible for the transport of all chemical, biological, radiological (nuclear) materials.

Biological warfare can also specifically target plants to destroy crops or defoliate vegetation. The United States and Britain discovered plant growth regulators (i.e., herbicides) during the Second World War, which were then used by the UK in the counterinsurgency operations of the Malayan Emergency. Inspired by the use in Malaysia, the US military effort in the Vietnam War included a mass dispersal of a variety of herbicides, famously Agent Orange, with the aim of destroying farmland and defoliating forests used as cover by the Viet Cong. Sri Lanka deployed military defoliants in its prosecution of the Eelam War against Tamil insurgents.

Anti-livestock

During World War I, German saboteurs used anthrax and glanders to sicken cavalry horses in U.S. and France, sheep in Romania, and livestock in Argentina intended for the Entente forces. One of these German saboteurs was Anton Dilger. Also, Germany itself became a victim of similar attacks – horses bound for Germany were infected with Burkholderia by French operatives in Switzerland.

During World War II, the U.S. and Canada secretly investigated the use of rinderpest, a highly lethal disease of cattle, as a bioweapon.

In the 1980s Soviet Ministry of Agriculture had successfully developed variants of foot-and-mouth disease, and rinderpest against cows, African swine fever for pigs, and psittacosis to kill the chicken. These agents were prepared to spray them down from tanks attached to airplanes over hundreds of miles. The secret program was code-named "Ecology".

During the Mau Mau Uprising in 1952, the poisonous latex of the African milk bush was used to kill cattle.

Defensive operations

Main article: Biodefense

Medical countermeasures

In 2010 at The Meeting of the States Parties to the Convention on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and Their Destruction in Geneva the sanitary epidemiological reconnaissance was suggested as well-tested means for enhancing the monitoring of infections and parasitic agents, for the practical implementation of the International Health Regulations (2005). The aim was to prevent and minimize the consequences of natural outbreaks of dangerous infectious diseases as well as the threat of alleged use of biological weapons against BTWC States Parties.

Public health and disease surveillance

It is important to note that most classical and modern biological weapons' pathogens can be obtained from a plant or an animal which is naturally infected.

In the largest biological weapons accident known—the anthrax outbreak in Sverdlovsk (now Yekaterinburg) in the Soviet Union in 1979—sheep became ill with anthrax as far as 200 kilometers from the release point of the organism from a military facility in the southeastern portion of the city and still off-limits to visitors today, (see Sverdlovsk Anthrax leak).

Thus, a robust surveillance system involving human clinicians and veterinarians may identify a bioweapons attack early in the course of an epidemic, permitting the prophylaxis of disease in the vast majority of people (and/or animals) exposed but not yet ill.

For example, in the case of anthrax, it is likely that by 24–36 hours after an attack, some small percentage of individuals (those with the compromised immune system or who had received a large dose of the organism due to proximity to the release point) will become ill with classical symptoms and signs (including a virtually unique chest X-ray finding, often recognized by public health officials if they receive timely reports). The incubation period for humans is estimated to be about 11.8 days to 12.1 days. This suggested period is the first model that is independently consistent with data from the largest known human outbreak. These projections refine previous estimates of the distribution of early-onset cases after a release and support a recommended 60-day course of prophylactic antibiotic treatment for individuals exposed to low doses of anthrax. By making these data available to local public health officials in real time, most models of anthrax epidemics indicate that more than 80% of an exposed population can receive antibiotic treatment before becoming symptomatic, and thus avoid the moderately high mortality of the disease.

Common epidemiological warnings

From most specific to least specific:

1. Single cause of a certain disease caused by an uncommon agent, with lack of an epidemiological explanation.
2. Unusual, rare, genetically engineered strain of an agent.
3. High morbidity and mortality rates in regards to patients with the same or similar symptoms.
4. Unusual presentation of the disease.
5. Unusual geographic or seasonal distribution.
6. Stable endemic disease, but with an unexplained increase in relevance.
7. Rare transmission (aerosols, food, water).
8. No illness presented in people who were/are not exposed to "common ventilation systems (have separate closed ventilation systems) when illness is seen in persons in close proximity who have a common ventilation system."
9. Different and unexplained diseases coexisting in the same patient without any other explanation.
10. Rare illness that affects a large, disparate population (respiratory disease might suggest the pathogen or agent was inhaled).
11. Illness is unusual for a certain population or age-group in which it takes presence.
12. Unusual trends of death and/or illness in animal populations, previous to or accompanying illness in humans.
13. Many affected reaching out for treatment at the same time.
14. Similar genetic makeup of agents in affected individuals.
15. Simultaneous collections of similar illness in non-contiguous areas, domestic, or foreign.
16. An abundance of cases of unexplained diseases and deaths.

Bioweapon identification

The goal of biodefense is to integrate the sustained efforts of the national and homeland security, medical, public health, intelligence, diplomatic, and law enforcement communities. Health care providers and public health officers are among the first lines of defense. In some countries private, local, and provincial (state) capabilities are being augmented by and coordinated with federal assets, to provide layered defenses against biological weapon attacks. During the first Gulf War the United Nations activated a biological and chemical response team, Task Force Scorpio, to respond to any potential use of weapons of mass destruction on civilians.

The traditional approach toward protecting agriculture, food, and water: focusing on the natural or unintentional introduction of a disease is being strengthened by focused efforts to address current and anticipated future biological weapons threats that may be deliberate, multiple, and repetitive.

The growing threat of biowarfare agents and bioterrorism has led to the development of specific field tools that perform on-the-spot analysis and identification of encountered suspect materials. One such technology, being developed by researchers from the Lawrence Livermore National Laboratory (LLNL), employs a "sandwich immunoassay", in which fluorescent dye-labeled antibodies aimed at specific pathogens are attached to silver and gold nanowires.

In the Netherlands, the company TNO has designed Bioaerosol Single Particle Recognition eQuipment (BiosparQ). This system would be implemented into the national response plan for bioweapon attacks in the Netherlands.

Researchers at Ben Gurion University in Israel are developing a different device called the BioPen, essentially a "Lab-in-a-Pen", which can detect known biological agents in under 20 minutes using an adaptation of the ELISA, a similar widely employed immunological technique, that in this case incorporates fiber optics.

List of programs, projects and sites by country

United States

Main article: United States biological weapons program

• Fort Detrick, Maryland
  

• Researchers working in Class III cabinets at the U.S. Army Biological Warfare Laboratories, Camp Detrick, Maryland (1940s).
  • U.S. Army Biological Warfare Laboratories (1943–69)
    • Building 470
    • One-Million-Liter Test Sphere
    • Operation Sea-Spray
    • Operation Whitecoat (1954–73)
  • U.S. entomological warfare program
    • Operation Big Itch
    • Operation Big Buzz
    • Operation Drop Kick
    • Operation May Day
• Project Bacchus
• Project Clear Vision
• Project SHAD
• Project 112
• Horn Island Testing Station
• Fort Terry
• Granite Peak Installation
• Vigo Ordnance Plant

United Kingdom

Main article: United Kingdom and weapons of mass destruction § Biological weapons

• Porton Down
• Gruinard Island
• Nancekuke
• Operation Vegetarian (1942–1944)
• Open-air field tests:
  • Operation Harness off Antigua, 1948–1950.
  • Operation Cauldron off Stornoway, 1952.
  • Operation Hesperus off Stornoway, 1953.
  • Operation Ozone off Nassau, 1954.
  • Operation Negation off Nassau, 1954–5.

Soviet Union and Russia

Main article: Soviet biological weapons program

• Biopreparat (18 labs and production centers)
  • Stepnogorsk Scientific and Technical Institute for Microbiology, Stepnogorsk, northern Kazakhstan
  • Institute of Ultra Pure Biochemical Preparations, Leningrad, a weaponized plague center
  • Vector State Research Center of Virology and Biotechnology (VECTOR), a weaponized smallpox center
  • Institute of Applied Biochemistry, Omutninsk
  • Kirov bioweapons production facility, Kirov, Kirov Oblast
  • Zagorsk smallpox production facility, Zagorsk
  • Berdsk bioweapons production facility, Berdsk
  • Bioweapons research facility, Obolensk
  • Sverdlovsk bioweapons production facility (Military Compound 19), Sverdlovsk, a weaponized anthrax center
• Institute of Virus Preparations
• Poison laboratory of the Soviet secret services
• Vozrozhdeniya
• Project Bonfire
• Project Factor

Japan

Main article: Special Research Units

 

U.S. authorities granted Unit 731 officials immunity from prosecution in return for access to their research.

• Unit 731
• Zhongma Fortress
• Kaimingjie germ weapon attack
• Khabarovsk War Crime Trials
• Epidemic Prevention and Water Purification Department

Iraq

Main articles: Iraqi biological weapons program and Iraq and weapons of mass destruction

• Al Hakum
• Salman Pak facility
• Al Manal facility

South Africa

Main article: South Africa and weapons of mass destruction § Biological and chemical weapons

• Project Coast
• Delta G Scientific Company
• Roodeplaat Research Laboratories
• Protechnik

Rhodesia

Main article: Rhodesia and weapons of mass destruction

Canada

• Grosse Isle, Quebec, site (1939–45) of research into anthrax and other agents
• Experimental Station Suffield, Suffield, Alberta

List of associated people

Bioweaponeers:

Includes scientists and administrators

• Shyh-Ching Lo
• Kanatjan Alibekov, known as Ken Alibek
• Ira Baldwin
• Wouter Basson
• Kurt Blome
• Eugen von Haagen
• Anton Dilger
• Paul Fildes
• Arthur Galston (unwittingly)
• Kurt Gutzeit
• Riley D. Housewright
• Shiro Ishii
• Elvin A. Kabat
• George W. Merck
• Frank Olson
• Vladimir Pasechnik
• William C. Patrick III
• Sergei Popov
• Theodor Rosebury
• Rihab Rashid Taha
• Prince Tsuneyoshi Takeda
• Huda Salih Mahdi Ammash
• Nassir al-Hindawi
• Erich Traub
• Auguste Trillat
• Baron Otto von Rosen
• Yujiro Wakamatsu
• Yazid Sufaat

Writers and activists:

• Daniel Barenblatt
• Leonard A. Cole
• Stephen Endicott
• Arthur Galston
• Jeanne Guillemin
• Edward Hagerman
• Sheldon H. Harris
• Nicholas D. Kristof
• Joshua Lederberg
• Matthew Meselson
• Toby Ord
• Richard Preston
• Ed Regis
• Mark Wheelis
• David Willman
• Aaron Henderson