Recipes for Recombining DNA. A History of Molecular Cloning: A Laboratory Manual

This month, we’re excited to collaborate with History of Knowledge to celebrate the upcoming conference, Learning by the Book: Manuals and Handbooks in the History of Knowledge. The five-day event takes place at Princeton in June and features a “blogged conference” to complement traditional panel presentations. For the next few Thursdays, the Recipes Project will cross-post selections from the conference (with RP readers noting  the extended length, in keeping with HoK posts). These features are  just a taste of more than thirty works produced for the conference, and readers are invited to read the full selection here. Enjoy!

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Angela N.H. Creager

Since Warren Weaver coined the term “molecular biology” in the late 1930s, technological innovation has driven the life sciences, from the analytical ultracentrifuge to high-throughput DNA sequencing. Within this long history, the invention of recombinant DNA techniques in the early 1970s proved to be especially pivotal. The ability to manipulate DNA consolidated the high-profile focus on molecular genetics, a trend underway since Watson and Crick’s double-helical model in 1953. But the ramifications of this technology extended far beyond investigating heredity itself. Biologists doing research on a wide variety of molecules, including enzymes, hormones, muscle proteins, RNAs, as well as chromosomal DNA, could harness genetic engineering to copy the gene that encoded their molecule of interest, from whatever organism they worked on, and put that copy in a bacterial cell, from which it might be expressed, purified, and characterized. Many life scientists who wanted to use recombinant DNA techniques were not trained in molecular biology. They sought technical know-how on their own in order to bring their labs into the vanguard of gene cloners. Manuals became a key part of this dissemination of expertise.

What did it mean to clone a gene? Simply put, cloning is copying, and a gene is usually copied onto a vector that can replicate in a cell, so that the copied gene can be propagated and studied. In seeking to make copies of genes and move them around from organism to organism, biologists were inspired by bacteria, whose ability to exchange genetic material had been recognized in 1946 by Joshua Lederberg and Edward Tatum. It turned out that there were numerous genetic units that enabled gene exchange in bacteria, including lysogenic viruses and fertility factors. In 1952 Lederberg christened the entities “plasmids.”

By the 1960s, researchers were using these naturally-occurring gene shuttles in microbes to identify, map, and characterize bacterial genes.[1] Unsurprisingly, many biologists were more interested in tracking genes found in humans and other “higher organisms” (eukaryotes—plants, animals, and fungi—as opposed to the one-celled prokaryotes, mostly bacteria). The discovery of bacterial restriction enzymes, which sever DNA strands at specific base-pair combinations, inspired molecular biologists to attempt to use these as microscopic scissors. In principle, if a researcher could identify and locate a particular eukaryotic gene, she could use a restriction enzyme to “cut” it out of chromosomal DNA and insert it into a circular bacterial plasmid (Figure 1). Cloning eukaryotic genes was an immensely difficult task, and several early attempts faltered. Other efforts did not go forward due to the potential public health hazards of placing genes from widely-studied tumor viruses into E. coli, a bacterium that usually inhabits the gut of humans. No one knew whether exposure to bacteria toting these tumor-associated genes could give people cancer.

Figure 1. Image and caption from Congress of the US, Office of Technology Assessment, Impacts of Applied Genetics: Micro-Organisms, Plants, Animals (Washington, DC: US Government Printing Office), 5. Public Domain.

In 1973, a group of scientists at UCSF and Stanford, led by Herbert Boyer and Stanley Cohen, succeeded in placing a copy of a frog gene (one that encoded ribosomal RNA) into a bacterial plasmid. Not only was the inserted gene on its plasmid vector taken up and replicated by E. coli, but also the foreign DNA was expressed into the corresponding product RNA. Their 1974 publication became the much-cited proof that genes from a higher organism could be cloned and expressed in a bacterium.

Few scientists, however, had the specialized materials with which to achieve such a feat. Richard Roberts at Cold Spring Harbor discovered and purified many of the restriction enzymes essential for this work. He recalls that “Summer visitors would stop by with a tube of their favorite DNA in their pocket, just to see if we had an enzyme that would convert it into some useful fragments.” Unable to persuade his own institution to start manufacturing and selling restriction enzymes, Roberts helped the newly-founded New England Biolabs corner this market. The first company catalog was issued in 1975; their enzymes became indispensable to the early gene cloners. Biologists who worked on bacteria were able to rapidly exploit these newly commercialized enzymes and customized plasmids, so that the cloning of genes from microbes took off.

However, cloning of genes from higher organisms remained in the hands of the experts who could make the difficult techniques work. In 1977, Shirley Tilghman and other members of Philip Leder’s group cloned the first mammalian genes from mice.[2] In addition to academic researchers, biotech entrepreneurs were keenly interested in cloning eukaryotic genes. Simply obtaining genetic material from higher organisms in a form that could be searched for a specific gene was a formidable challenge. Tom Maniatis, part of the group that cloned the first human gene, created a human genomic “library” and shared it with other biologists.[3] But researchers also needed protocols and know-how. Courses (for practitioners, not only university students) became a popular way to meet this demand.

Cold Spring Harbor Laboratory had been offering summer courses on new laboratory techniques since the 1940s. One popular course, “Advanced Bacterial Genetics,” already offered researchers a chance to learn how to identify, map, and copy genes from prokaryotes. In 1980, Cold Spring Harbor Laboratory (CSHL) began offering a postgraduate summer course called “Molecular Cloning of Eukaryotic Genes.” James Watson, director of CSHL, asked Maniatis to teach this course, and others joined the effort. Nancy Hopkins, who had taught a tumor virology course that had just ended, stayed on for the cloning course. Ed Fritsch, a postdoc in Maniatis’s lab, put together the laboratory materials, and Helen Donis-Keller and Catherine O’Connell served as course assistants.[4]

The coursebook was made up of “consensus protocols” defining the field at the time (many of which were already circulating informally).[5] Upon advertising the postgraduate training course, “Molecular Cloning of Eukaryotic Genes,” more than 300 applied to take it. Only sixteen students could enroll. Watson immediately saw the opportunity to make cloning know-how available to a wider base of users through publication. Issuing an instructional guide from Cold Spring Harbor Laboratory would further consolidate the institution’s reputation for being at the vanguard of molecular biology—and there was already a tradition there of publishing course manuals as books.

Figure 2. Cover of Tom Maniatis, Ed Fritsch, and Joe Sambrook, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1982). Author photo.

Watson wanted Maniatis on the team of authors, as his reputation in cloning genes was already formidable. But he had recently moved to Caltech, where he was busy chairing an NIH study section and running his own lab. He only agreed to prepare a manual based on the course if he had significant help.[6] Watson persuaded Joe Sambrook, a long-time tumor virologist at the lab, to join the effort. Although Sambrook had not taught the summer course, he did have extensive relevant knowledge, and he would do a lion’s share of the manual-writing.[7] Fritsch, who was about to leave for a tenure-track faculty position at Michigan State University, remained involved with the project having helped teach the course twice.[8] In the end, the collaboration was productive, and the first edition was published in 1982 (Figure 2). Maniatis handed off teaching of the “Molecular Cloning of Eukaryotic Genes” summer course at CSHL to others the same year as the manual came out.

The three authors explain in the Preface that because “the manual was originally written to serve as a guide to those who had little experience in molecular cloning, it contains much basic material.”[9] Indeed, the book was full of both recipes and tips. That said, part of its success, according to one early user, was that it communicated enough about the science behind the recipes that users were able to trouble-shoot the problems they ran into.[10] And part of the utility of the book was that, by virtue of its plastic-ring binding, it could be laid flat on a laboratory bench [11] (Figure 3).

Figure 3. Pages 92 and 93 of Maniatis, Fritsch, and Sambrook, Molecular Cloning: A Laboratory Manual. One can see how the book is spiral bound so it lays flat when open. Author photo.

 

Just as Watson had suspected, Molecular Cloning met widespread demand. There were orders for more than 5000 copies before the publication date. Consequently, the press sold 5113 copies the first month of its appearance, in July 1982 (as compared with its original number for sales projected by the press: 210 copies). In August 988 copies were sold, in September 2487, in October 1863, and in November 768. That fall, Molecular Cloning was outselling every other book in the press’s line-up.[12] As a reviewer for the British Society for Developmental Biology put it, “no laboratory with any serious interest in molecular biology of development and their [sic] cloning should be without it.”[13] By late June 1983, more than 18,000 copies had been sold.[14] Plans for a second edition, initially scheduled for 1984, were already underway.[15] The second edition, which actually appeared in 1989, was received just as enthusiastically as the first. As a reviewer in Nature put it,

Few molecular biologists welcome publication of any of the many protocol books that promise to be the single source for their laboratory methods. For the most part, such laboratory methods fall far short of this goal. So why the excitement surrounding the long-awaited second edition of the classic guide, Molecular Cloning, which first appeared in 1982? The original version immediately filled the need for an anthology of laboratory procedures pertinent to the emerging field of recombinant DNA. With the 545-page spiral-bound paperback in hand, virtually any experimentalist could make a stab at cloning and have a reasonable expectation of success.[16]

Figure 4. Frederick M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J. G. Seidman, John A. Smith, and Kevin Struhl, eds., Current Protocols in Molecular Biology, vol. 1 (New York: John Wiley & Sons, 1987). Author photo.

In short, the Cold Spring Harbor Laboratory publication became the canonical manual—or “Bible”—for gene cloners. Extending this common metaphor, one biochemist made reference to “those who daily workshop the Cold Spring Harbor idol.”[17] But the deity had rivals. Its strongest competitor was Current Protocols in Molecular Biology, introduced in 1987 by a group of researchers based at Massachusetts General Hospital.[18] Sarah Greene was the original publisher, but the series was soon bought by Wiley. Rather than being written by three authors, this manual was produced by an entire team of scientists, who contributed individual pieces on various techniques. In addition, Current Protocols had a very different way of dealing with the rapid growth (and obsolescence) of techniques—the book was designed to be expanded via subscription. Through a quarterly update service, subscribers received supplements to insert into the original loose-leaf binder, which was separated into sections by preprinted dividers (Figures 4 and 5). This meant that the Table of Contents also needed frequent updating. Five thick binders were published in the original series (Figure 6).

Figure 5. Ausubel et al., eds., Current Protocols in Molecular Biology, open so that dividers between the sections of the loose-leaf bound book are visible. Author photo.

The loose-leaf format proved unwieldy, and in 1989 Wiley published Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology. This single volume work was bound as a traditional text, with wide pages in a format that would prop open easily on the back of a lab bench. The challenge of updating was more easily accommodated by the growth of multimedia technologies in the 1990s. The 2001 edition came with a CD-ROM “Lab Book.” By the third edition (2001), Molecular Cloning: A Laboratory Manual also had an associated website for its publication. Moving manuals online put knowledge at one’s fingertips in a new way, yet the demand for guides that can be plopped open on a lab bench has meant that print versions retain value, as evidenced by the publication of a fourth edition of Molecular Cloning in 2012. Most fields of life science today, including bioinformatics, cell biology, immunology, neuroscience, stem cell science, and toxicology, have their go-to manuals and protocol books, in print and online.

Figure 6. Three of the first five volumes, published in the late 1980s, of Ausubel et al., eds., Current Protocols in Molecular Biology, stacked on office table. Author photo.

These “cookbooks” occupy the shelves, benches, and hard-drives of most biology labs, important if unnoticed. Their ubiquity enriches our understanding of the scientific process. An obsession with innovation may blind us to the importance of procedure, repeatability, and tried-and-true methods. Manuals make discovery possible, by leading scientists through the routine steps of their experiments and (if the manual is good) helping them trouble-shoot when experiments fail. In a world of hyper-specialized research, guide books are bridges, carrying technical know-how between laboratories and enabling researchers to master the latest methods without going back to school.

 

[1] For an overview see William Hayes, The Genetics of Bacteria and their Viruses (New York: John Wiley & Sons, 1965).

[2] S. M. Tilghman, D. C. Tiermeier, F. Polsky, M. H. Edgell, J. G. Seidman, A. Leder, L. W. Enquist, B. Norman, and P. Leder, “Cloning Specific Segments of the Mammalian Genome: Bacteriophage  Lambda Containing Mouse Globin and Surrounding Gene Sequences,” Proceedings of the National Academy of Sciences, USA 74 (1977): 4406–4410; D. C. Tiermeier, S. M. Tilghman, and P. Leder, “Purification and Cloning of a Mouse Ribosomal Gene Fragment in Coliphage Lambda,” Gene 2 (1977): 173–191.

[3] Richard M. Lawn, Edward F. Fritsch, Richard C. Parker, Geoffrey Blake, and Tom Maniatis, “The Isolation and Characterization of Linked d- and b-Globin Genes from a Cloned Library of Human DNA,” Cell 15 (1978): 1157–1174.

[4] Interview with Tom Maniatis, Columbia University, New York, Tuesday, Oct. 25, 2016.

[5] Jonathan Karn, “Yet Another Maniatis?” Trends in Genetics 4/9 (Sept 1988): 268.

[6] He was chair of an NIH study section and running a big lab, which involved constantly writing grants, as well as teaching a full load at Caltech. Interview with Maniatis, op. cit.

[7] Joe Sambrook was a talented and combative British tumor virologist whom Maniatis met when doing his cloning work at CSHL in the 1970s. Involving him as an author of the molecular cloning manual enabled a certain redress at CSHL. A few years earlier Sambrook had contributed significantly to John Tooze’s Tumor Virology book, but this was not acknowledged by his being an author. Personal communication, Alex Gann, 26 May 2010.

[8] Interview with Maniatis, op. cit.

[9] Tom Maniatis, Ed Fritsch, and Joe Sambrook, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1982), iii.

[10] Conversation with Michael S. Levine, fall 2016.

[11] Stephanie Radner, Yong Li, Mary Manglapus, and William J. Brunken, “Joy of Cloning: Updated Recipes,” Trends in Neuroscience 25/11 (Nov 2002): 594–595.

[12] Memorandum from Susan Gensel to Jim Watson, 10 Dec 1982, re: sales at the American Society for Cell Biology meeting, Watson papers, Cold Spring Harbor Laboratory Archives. At that meeting Molecular Cloning sold 83 copies, and all the other sales together, 22 titles in all, made up 102 copies.

[13] British Society for Developmental Biology Newsletter VII, October 1982, review of Molecular Cloning: A Laboratory Manual, copy in Cold Spring Harbor Laboratory Archives.

[14] Cold Spring Harbor Laboratory Annual Report 1982, 12.

[15] J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989). This edition was three volumes.

[16] Stuart Orkin, “By the Book,” Nature 343 (15 Feb 1990): 604–605, on 604.

[17] S. J. W. Busby, “Comprehensive Cloning,” Trends in Genetics 4/12 (Dec 1988): 352.

[18] The Harvard-affiliated editors were Frederick Ausabel, Robert Kingston, Jonathan Seidman, and Kevin Struhl.

Blog Series: Learning by the Book

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Making Senses: Artisanal Practice and Sensory Perception in an Early Modern French Manuscript

By Tillmann Taape

Ms Fr. 640 was written in French by an unknown craftsperson in Toulouse, likely between 1580 and 1600. [1] It is an intriguing and eclectic source, with entries ranging from medical recipes to metalwork and pigment-making, and it forms the core of the Making and Knowing Project at Columbia University, introduced previously on the Recipes Blog in a post by our Director, Pamela Smith.

With its numerous instructions for making things, our manuscript provides a rich case study for the way artisans worked with and thought about materials. As previous posts in this series on Recipes and the Senses have shown, physicians, alchemists, apothecaries, and other craftsmen recognised in their bodies and its senses an important set of tools for understanding and manipulating the material world, and historians pay increasing attention to these embodied and sensory ways of knowing. In this post, I will share a few examples of the rich language of the senses in Ms. Fr. 640. As one might expect from a manuscript including painting and sculpture, the eye often takes precedence over the other senses. However, a discussion of the visual in the manuscript would by itself be far beyond the scope of a single post – we spent much of this year just trying to figure out how the author-practitioner conceptualises different pigments and shades of blue. The aim here, therefore, is to focus on the oft-neglected non-visual senses and what they can teach us about our author-practitioner’s concept of the material world, his ‘material imaginary’.

Smell

A strong smell was often a sign that things had gone wrong – the papier-mâché had turned rotten while being left to soak, or a kitchen pot had been made with too much latten (a copper alloy), which ‘stinks and smells bad’ (fol. 36v). However, smells could also help identify the materials needed for a recipe. The ingredient list for a metal alloy, for example, includes the intriguingly specific ‘congealed mercury with the smell of tin’ (fol. 92v). Musing on one of his favourite topics, the properties of fine sand used for metal casting, the author-practitioner notes that

white sand smells like sulphur when heated, and I believe it would melt. And as the substance has been cast in it, it acquires in the mold a lustre as if it were leaded or vitrified. I believe that glassmakers could use it (fol. 99r).

In addition to his observation of a vitreous glaze on the cast object, it is the sulphurous smell which suggests to the author-practitioner that this particular kind of sand is prone to melt and could even be used for making glass. Throughout the manuscript, sulphur does indeed appear as a material which can easily be melted and used to cast small objects, and even appears to function as a sort of material metaphor for transformation and experimentation.

Listen

The sense of hearing becomes itself the subject of a short entry. Under the heading ‘hearing from afar’, the author-practitioner records one of the tidbits of advice and tricks for daily life which are scattered here and there throughout the manuscript: ‘Make a small hole in the ground, put your ear against it during the night or during a quiet time, and you will easily hear muffled sounds’ (fol. 125r). In addition to facilitating amateur espionage, specific noises could serve as helpful indicators in the workshop. Before casting metal into a mould made from cuttlefish bone, the author-practitioner writes, one has to make sure that it is completely dry: ‘you will know that they are dry enough when, after having held them near the fire a little, their inside and the impression scream & crackle when you hold them up to your ear’ (fol. 145r). If one was prepared to listen carefully, the materials themselves could tell when they were ready to be worked upon.

Cuttlefish bone used for casting metal objects. © The Making and Knowing Project

Taste

The sense of taste could also help to assess and adjust one’s materials. To make ‘essence of sal ammoniac’, for example, ‘the size of two chestnuts of pulverized sal ammoniac suffices in a pot of water, and to the tongue you find the water moderately salty, for too much is not good’ (fol. 111v). The concentration of the sal ammoniac solution was clearly of some importance here, and like in most early modern recipes, the given measurements – size of a chestnut, a pot of water – might not yield very consistent results, so a qualitative sensory indication – ‘moderately salty’ – is added as a further point of reference. As well as checking one’s own procedures, taste could of course be used to assess the quality of merchandise. The city of Toulouse, where our manuscript was compiled, gained much of its considerable wealth from the trade in woad, a blue dyestuff whose French name, pastel, is a likely origin of the term ‘pastel’ colours in English and other European languages. It is not surprising, therefore, that the author-practitioner mentions this sought-after material and tells us how to tell the good from the bad. This involves several steps, including inspection and a dyeing test, but the first step is a taste test: ‘The goodness of the woad is known when, put in the mouth, it gives a taste as of vinegar’ (fol. 39r).

Touch

Perhaps unsurprisingly for someone who clearly worked with his hands a lot, the sense of touch plays a particularly important and intriguing part in the author-practitioner’s practice and writing. Returning to his favourite topic – the different kinds of sand or plaster used for casting moulds – he describes how the addition of a substance called alum de plume (literally ‘feather alum’ – it probably refers to a group of minerals known as feldspars in English) helps the mould hold together because it forms fibrous structures (hence probably the reference to feathers). Its production requires a complex process of heating and grinding up in a mortar. In the margin next to the recipe, the author-practitioner notes that one should grind the alum slowly and in small portions, and finally ‘render it very fine & soft to the touch’ (fol. 108v). The manuscript is full of these kinds of haptic properties to indicate the appropriate consistency or particle size of materials. Another ‘sand’ for casting, for example, is made with ‘the bone of oxen feet, very burned & pulverized & ground on porphyry, until it is not felt between your fingers’ (fol. 84v). Intriguingly, here the reader is told to stop grinding not when they can feel a particular sensation, but when they can no longer feel the material at all with their fingers.

As it turns out, this criterion of eluding the sense of touch was an important technical concept for early modern artisans. Our former Making and Knowing postdoc Jenny Boulboullé and former students, Raymond Carlson and Jordan Katz, have shown that the term impalpable, that is to say ‘un-feelable’ or ‘impalpable’, is central to the way the author practitioner experiences and thinks about different kinds of materials used for casting moulds.[2] Furthermore, they found that he is not the only one: the use of the term ‘impalpable’ is used in published works on metallurgy, such well-known book Pirotechnia by the sixteenth-century Italian founder and metallurgist Vanoccio Biringuccio, and the Secreti, a famous book of secrets attributed to Alessio Piemontese. In his emphasis on the haptic sensation of a material being impalpable, then, the author-practitioner speaks to a sensory terminology apparently widely shared by expert makers.

‘Knead as if you wanted to make bread’: making stucco in the Making and Knowing Lab. © The Making and Knowing Project

Describing specific sensory experiences can be difficult, and it makes sense to refer to well-known parallels from daily life – a smell like sulphur, a taste like vinegar, and so on. When it comes to the sense of touch, too, the author-practitioner relates processes described in his recipes to everyday practices. As our former student Emma Le Pouésard has shown, the practices surrounding making bread were a particularly fruitful source of these kinds of comparisons.[3] To unmould a cast object, one should ‘strongly separate the moulds as if you wanted to tear bread apart’ (fol. 114v). In an age before thermostats, this could even provide a way of gauging consistent temperatures. For one’s domestic taxidermy needs, the author-practitioner writes, one could dry animals ‘in an oven as warm as when bread has been taken out’ (fol. 129v). In a recipe for making stucco, bread making is used as a referent for working up the right kind of consistency: the recipe tells us to ‘knead as if you wanted to make bread’, until the stucco paste is ‘firm as bread dough that is ready for the oven’ (fol. 29r).

When we tried making stucco in the Making and Knowing Lab in the Fall semester, this proved to be very useful guidance. While we were not experienced bakers in the way that many early modern householders probably were, we could draw on our experience from one of our ‘skillbuilding’ exercises a few weeks earlier, when we made bread to use as a mould for wax casting, replicating one of the most intriguing processes in the manuscript. When it came to making stucco and mixing the right amounts of tragacanth gum and rye flour or champagne chalk, the author-practitioner’s instructions about kneading to a consistency like bread dough were very useful, especially in the absence of any other indication of measurements. By adding flour until we achieved a dough-like mass which would ‘stretch enough without breaking’ (fol. 29r), we eventually produced stucco which displayed fine detail and could be detached from the mould without too much trouble.

Even this brief tour of Ms. Fr. 640 shows that much is to be gained by paying attention to the non-visual senses in recipes and practical instructions. In the absence of precise standardised measurements and procedures, sensory descriptions were paramount to articulating a material’s properties, whether it was of good quality, or how much longer it needed to dry, boil, soak, or be crushed in a mortar.

 

[1] High-res digital images of BNF Ms. Fr. 640 are available through Gallica. The Making and Knowing Project is preparing a Digital Critical Edition of the Manuscript. In the meantime, readers may wish to refer to our Minimal Edition prototype (with translation still in progress).

[2] Raymond Carlson and Jordan Katz, ‘Casting in a Box Mold’, The Making and Knowing Project, A Digital Critical Edition of BnF Ms Fr. 640, forthcoming. For more information see http://www.makingandknowing.org/.

[3] Emma Le Pouésard, ‘Pain, Ostie, Rostie: Bread in Early Modern Europe’, The Making and Knowing Project, A Digital Critical Edition of BnF Ms Fr. 640, forthcoming. For more information see http://www.makingandknowing.org/.