The Genetic Code to Synthetic Biology: Reminiscence on the Life and Legacy of a Visionary Scientist Professor Har Gobind Khorana Lecture by Shiladitya DasSarma, Professor in the University of Maryland and Ph.D. student of Prof. Khorana on the occasion of the Centenary of the Birth of the Nobel laureate, on January 9 and 10, 2022, upon the invitation of the Indian Society of Human Genetics and Council of Scientific and Industrial Research. The CSIR oration is also available on YouTube. Watch Here on You Tube or Click Below to Listen to the Lecture and Scroll Down for Slides and Transcription ©Shiladitya DasSarma
	Slide #1: I will be speaking today about Professor Har Gobind Khorana's work on the genetic code and synthetic biology and in particular I would like to reminisce on the life and scientific legacy of the visionary and inspirational scientist on the occasion of his birth centenary. I was privileged to be a graduate student in his laboratory from 1979-1984,with support from a graduate fellowship from the US National Science Foundation.
	Slide #2: Professor Khorana had a very long and distinguished career spanning six decades, starting in 1952 and going all the way until 2007, when he was in his mid-eighties. He had many remarkable achievements during the course of his life. He synthesized a very complicated nucleotide cofactor coenzyme A. He also pioneered the synthesis of DNA oligonucleotides and polynucleotides of defined sequence, which allowed him to decipher the genetic code. He went on to do the first complete synthesis of a gene, including a functional gene. He then transitioned to membrane biology, where he determined the mechanism of light-driven transmembrane ion translocation, and opened the field of archaeal genetics. Finally over the last 20 years of his career, he pioneered studies of the mammalian visual system and signal transduction.
	Slide #3: He won the Nobel prize in medicine for deciphering the genetic code in 1968, which is one of the greatest achievements of 20th century science, at the age of 46. His Nobel Prize medal can be seen on this slide.
	Slide #4: In addition, Dr. Khorana was recognized during his life with many other awards. He was elected a member of the US National Academy of Sciences, he won the Horowitz prize, and was awarded the Lasker award. He was also recognized by awarding of the National Medal of Science by the President of the United States. He also won many awards in India: the Padma Presidential award, the JC Bose medal, and he was also elected a foreign member of the Indian Academy of Sciences. In addition he also was recognized in many other countries including Canada, the United Kingdom, Germany, Italy, Japan, and the former Soviet Union.
	Slide #5: I was privileged to be a graduate student with Professor Khorana in MIT from 1979 to 1984, during a time when the laboratory was transitioning from nucleic acids research to membrane biology. It was a time of great excitement, and after the first part of my talk where I provide a brief introduction to his education and career, I would like to provide some personal reminiscences and reflections on my time in the Khorana lab. Next, I would like to highlight his major accomplishments in science, and then finally close with how he inspired his students and his scientific legacy.
	Slide #6: Khorana was born in a small village in Punjab in early January, 1922, a hundred years ago. His father Sri Ganput Rai was a tax collector and his mother was Srimati Krishna Devi Khorana. He was the youngest of five children, with three elder brothers and one elder sister. He received his primary education in the village, and for high school, he went to a nearby city. He excelled at his studies in school.
	Slide #7: He won a scholarship to attend Punjab University in pre-independence India, where he earned a B.Sc. degree, with honors, first class, in chemistry in 1943 and a M.Sc. also with honors, first class with Professor Mohan Singh as mentor. This was a very turbulent time in history. World War II had been ongoing between 1939 and 1945, and it was the lead-up to the Indian independence, which was won in 1947.
	Slide #8: But Khorana was a very focused student and he was able to win a scholarship to study abroad in the UK through the Department of Agriculture, Government of India. He traveled to the UK at the end of World War II and studied organic chemistry at the University of Liverpool in the laboratory of Dr Alexander Robertson, a Fellow of the Royal Society. After three years, he earned a PhD in the field of alkaloids and other natural products. The structure shown is for violacein, which is a plant alkaloid, with an aromatic ring structure with a nitrogen atom, not unlike that of bases in nucleic acids.
	Slide #9: After his PhD, he went to Zurich, Switzerland, still on his Indian scholarship, and worked with Vladimir Prelog at the Swiss Federal Institute of Technology for 11 months. After this time he returned back to India. This was in the immediate aftermath of the Indian independence, and unfortunately, he was not able to find employment in India. So he returned back to the UK, this time with a British Nuffield Fellowship, to Cambridge University to work as a postdoctoral fellow with Sir Alexander Todd, whose research area was nucleotides and nucleotide coenzymes.
	Slide #10: In 1952, he had the good fortune of meeting Gordon Strum from British Columbia, Canada. Strum had come to Cambridge to recruit an organic chemist for a research institute, the British Columbia Research Council (or BCRC) in Canada. Strum interviewed Professor Khorana and offered him an independent research position, which Professor Khorana accepted. By then, he had married Esther Sibler, a Swiss citizen whom he had met when he was in Zurich. During the 1950s, they went on to have a family of three children in Canada. After eight years at the BCRC, in 1960, Dr Khorana moved as Professor and Co-Director to the Institute of Enzyme Research at the University of Wisconsin, Madison in the United States. Ten years later, in 1970, he again moved, this time to take a position as professor of chemistry and biology at the Massachusetts Institute of Technology, MIT. He remained in MIT until his retirement in 2007.
	Slide #11: The MIT campus, on the banks of the Charles River across from Boston, seen in this aerial photo, shows a large complex of interconnected buildings, reflecting the both the connections between disciplines and the highly interdisciplinary nature of the institution. Among the interconnected buildings were the biology and chemistry buildings, connected via a five story walking bridge. Dr Khorana’s lab and office was in the chemistry building, around the corner from the biology headquarters.
	Slide #12: I had my first meeting with Professor Khorana in the Spring of 1979 in the chemistry building, which was referred to by its number, Building 18. I had been admitted into the chemistry graduate program and I was visiting as a prospective graduate student. I discovered that Dr Khorana had a very large laboratory, occupying the entire 5th floor. Khorana worked together with Uttam Raj Bhandary, a former postdoctoral associate in his Wisconsin lab, who was now a faculty member in the MIT biology department. Their two laboratories worked together on many projects, including the project I would work on for my Ph.D.
	Slide #13: In the first meeting with Dr Khorana in his office, I asked him about his current research. We initially discussed earlier work on the total synthesis of two genes, the alanine tRNA gene from yeast and tyrosine suppressor tRNA gene from E. coli. The yeast tRNA gene had been published in an article in Nature, plus13 papers which were published together in a single issue of the Journal of Molecular Biology in 1972. In 1979, 17 papers were published from his lab reporting the total synthesis of the tyrosine suppressor transfer RNA in the Journal of Biological Chemistry. He then explained that his laboratory had recently changed its focus to membrane proteins. He also told me about recent work, not yet published, on the purple membrane protein of the Archaeon, named Halobacterium halobium. The amino acid sequence of the purple membrane protein, bacteriorhodopsin, was being determined and the cloning of its gene was of interest. This would be the project I would work on if I joined the group. I soon decided that I would join his group, and that of RajBhandary’s, through the biology department, which offered the PhD in biochemistry at MIT.
	Slide #14: As I joined the group, I became a part of some wonderful traditions, one of which was an annual group photo. Shown is the group photo from 1983, with Dr Khorana standing at the center and Dr. RajBhandary to his left. Most of the group members were postdoctoral associates. There were only two graduate students in Dr. Khorana’s lab at this time, including myself. I'm the fellow who can be seen kneeling at the left side of the group photo.
	Slide#15: Another tradition in the group was regular group meetings. In fact Dr Khorana had meetings nearly every day of the week - including Saturday. The group meetings started Monday morning, with a three-hour session from 9am to noon, when two group members would present their research results from the last few months. One of the requirements for these meetings was submission of a written lab report on Friday, prior to Monday, so that Dr Khorana would have a chance to review the work in detail over the weekend. Another memorable meeting was the Thursday afternoon “tea club”. Dr Khorana made tea for the group using a giant Erlenmeyer flask and provided cookies. This was a more informal meeting where group members would present their latest findings. Soon after I started, he also established the bacteriorhodopsin cloning and expression group, which met on Saturday mornings, and to entice people to come into the laboratory early Saturday, he brought doughnuts. In addition to all the work meetings, he would also invite group members to leisure activities, such as picnics at his New Hampshire cottage, and occasionally to his home for holiday dinners.
	Slide #16: Dr Khorana enjoyed going to scientific meetings immensely and encouraged his students to also attend. In fact, the very first summer after I joined, he arranged admission for me to attend a Gordon research conference. These are small conferences that are held in boarding schools and small colleges just north of Boston, usually in New Hampshire, which are devoted to specific topics and organized by leaders in the field. I remember going to two GRCs with Dr. Khorana, on nucleic acids in 1980 and in 1982. On the left, you can see a group picture from 1982, where Dr Khorana is standing near the back row and I'm standing near the front, on the right. Khorana also went to a number of international meetings, including several in India. The 1983 International Congress on Genetics in New Delhi was one which both he and I attended, and that was quite a wonderful experience.
	Slide #17: In addition, Professor Khorana had many, many visitors. Among them were eminent scientists who would visit his lab, and I remember being a part of some exciting scientific discussions. One of my memories from these small meetings is of a wood block on a side table in his office. On top of that wood block was written: “Secret to success” and a great temptation to lift the top of wood block to find the answer. When I opened it, what did it say? “Hard work!” This, I think, really epitomized Dr Khorana's philosophy.
	Slide #18: During the five years I spent in his laboratory, I co-authored three papers in the prestigious Proceedings of the National Academy of Sciences of the USA, in 1982, ‘83, and ‘84. These papers were co-written with Professors Khorana and RajBhandary. The three of us drafted these papers together, primarily by hand or using a typewriter, as common in the pre-word processing and personal computer days. I can remember that Dr Khorana, in particular, liked to revise extensively, cutting and pasting to move the passages around, and in these cases, he would literally cut selected passages out with scissors and paste them with scotch tape into a different place.
	Slide #19: By the Spring of 1984, I had completed my thesis work, and Dr Khorana gave me the go-ahead to write my thesis. I successfully defended my thesis on April 27^th. The snapshot seen, taken with a Polaroid camera, remains a favorite of mine, taken after my PhD defense at a small party he organized in the chemistry building. And my thesis title page with Professor Khorana’s signature is shown on the right.
	Slide #20: Next, I’d like to move on to his major accomplishments in science. I want to begin with this favorite quote from Dr. Khorana: "If you are doing research in both chemistry and biology, you're involved in a lot of excitement…and that does not leave one with much time for anything else." This quote epitomized Dr. Khorana’s philosophy of life. This was the reason he could conduct groundbreaking research over six decades. Another aspect of his approach was that he assembled and directed large teams of scientists. Even back in the 1950s, this photograph showed that his group at the BCRC was large. One group member standing next to Dr. Khorana at the extreme left is Michael Smith, who also went on to win the Nobel prize.
	Slide #21: The area of research that Professor Khorana focused on initially was the development of tools for nucleic acid synthesis. Since nucleic acids are long polymers which require iterative joining of nucleotides, yields from each step must be as high as possible in order to have a final yield good enough for the synthesis to be successful. Side reactions for each step must be minimized to avoid unwanted by-products. Early on, Dr. Khorana made a decision to use a little known condensing agent, DCC or dicyclohexylcarbodiimide. This condensing agent was extremely valuable in his work on nucleic acid synthesis. No wonder, Dr Khorana also said:“If you want to break new ground in science, you have to walk the path alone."
	Slide #22: A synthetic reaction that is promoted by DCC is the condensation of two deoxyribonucleotide bases into a dinucleotide. The dinucleotide can be condensed into a trinucleotide, again using DCC, or a tetranucleotide, as shown on the left. On the right is shown several protecting groups which were used, and they may be successively added and removed, as necessary, in order to direct the synthesis of the appropriate deoxyribo-oligonucleotide sequence desired. The 5’-end could be protected using a methoxytrityl group, which can be removed using acid, and the 3’-end could be protected by an acetyl group, which can be removed using base. And nucleotide base amines could be protected using para-methoxybenzoate. So this was this general approach used for synthesis of deoxyribo-oligonucleotides.
	Slide #23: The crowning achievement during this time was the synthesis of Coenzyme A, which is one of the most complex nucleotide cofactor or coenzyme that exists. And it also plays a central role in the metabolism of fatty acids and the TCA cycle. Nearly five percent of all enzymes in the cell utilize Coenzyme A. The cofactor has three different components. As you can see on the right hand side Coenzyme A is composed of a phosphorylated adenosine, pantothenic acid and aminoethanethiol. The two final steps, in a very complicated synthetic pathway for synthesis of Coenzyme A, require DCC for condensations. In the final step, condensation produces phosphoric anhydride under anhydrous conditions, and in the previous step, a cyclic phosphodiester with a protecting group was also produced using DCC. With the successful synthesis of Coenzyme A, Professor Khorana moved his group to Wisconsin.
	Slide #24: During the 1950s, there had been many seminal discoveries in the field of biochemistry and genetics, including: double helical DNA structure and base pairing, and the requirement of ribosomes for protein synthesis as well as the crucial roles for messenger RNAs and transfer RNAs. These discoveries led to the central dogma of molecular biology, that information flows from DNA to RNA to protein. And by the early 1960s it was clear that there was colinearity between DNA and polypeptides, through genetic and biochemical experiments. However, since DNA could not be sequenced yet, the underlying code was not know.
	Slide #25: It was not clear how the sequence of A's, C's, G's and T's in DNA transcribed into messenger RNA was translated into the string of amino acids in proteins. To unravel this mystery, polynucleotides of defined or known sequence were required. And Professor Khorana's lab was able to synthesize dinucleotides, trinucleotides and tetranucleotides of almost any sequence, and in double-stranded form. His lab was the only lab that could readily synthesize them at that time. And in addition, he was able to take these short oligonucleotides and polymerize them into repeating DNA polymers and then transcribe them into RNA polymers.
	Slide #26: The polymerization of short repeated sequences was accomplished through multiple rounds of enzymatic synthesis using DNA polymerase and deoxyribonucleotide triphosphates. In the first example, two homopolymers are seen, poly dT and poly dA, which are complimentary and can hybridize. With addition of DNA polymerase with dTTP and dATP, a DNA polymer is produced, with one strand of poly dA, base-paired to a strand of poly dT. Repeating dinucleotides, for example, dTG and dCA, could hybridized or base pair, and after extension through multiple rounds, produce poly dTG-dCA. And similarly, the Khorana lab could make repeating trinucleotide polymers, for example, poly dTTC-dGAA, and tetranucleotide repeat polymers, poly dTATC-dGATA.
	Slide #27: After checking the fidelity of DNA synthesis by nearest neighbor analysis, Dr. Khorana could then use those DNA polymers for transcription into RNA with RNA polymerase and ribonucleotide triphosphates. RNA polymerase could transcribe either strand, depending on which nucleotides were added. In the first example with poly dTG:dCA, if A and C were added, poly CA would be obtained if U and G were added, poly UG would be obtained. Similarly with the trinucleotide, and tetranucleotide DNA polymers, if CTP was omitted, he would obtain poly GUA or poly GAUA, or if GTP was omitted, he would get poly UAC or poly UAUC. A wide variety of messenger RNA polymers were produced in this way and used for deciphering the genetic code.
	Slide #28: Once he had defined polynucleotides to use as messenger RNAs, he could then proceed with in vitro translation and determine which peptides were coded. For example, with polyCA, he obtained polypeptides with repeating two amino acids. In the first case, he obtained a repeating polymer of threonine and histidine. In the second case, it was a polymer of cysteine and valine. When he used the poly-trinucleotide polymers, for example, poly GUA he obtained polyvaline and polyserine. In the case of poly UAC, he obtained three different polymers: poly-tyrosine, poly-threonine and poly-leucine. And in the case of the tetranucleotides, he sometimes obtained short di- or tri- peptides. In the case of poly GAUA, he obtained a dipeptide containing aspartic acid and isoleucine, or in the case of poly AUAC, he obtained a polymer of four amino acids: tyrosine, isoleucine, serine and leucine. Taking all of this together, he was able to conclude that the genetic code was a triplet code with considerable redundancy. Most amino acids were encoded by more than one codon, the exceptions being methionine and tryptophan, which were coded by single codons. Methionine was usually coded by an AUG, although in some cases with GUG, when this valine codon constituted the start site for translation. And he was also able to tell which codons were stop codons and there were three of those, UGA, UAG, and UAA.
	Slide #29: By 1966, all of the available information was assembled together and the genetic code table became complete. This work included the contributions from many people, including Robert Holley and Marshall Nirenberg, who shared the Nobel Prize with Dr. Khorana in 1968. It was an amazing scientific feat, indeed, accomplished in just a few years: the genetic code. In addition, scientists would go on to show that translation systems from all different types of organisms: Bacteria, Archaea, Plants and Animals, all had the same "universal" or standard genetic code. These were profound discoveries with enormous implications for biology, for chemistry, and for evolution. The near universality of the genetic code is the strongest evidence that all life on Earth originated from a common ancestor.
	Slide #30: Soon after the genetic code was complete, the Khorana lab moved on to the next project, which was complete synthesis of a gene. Because genes are much longer, it was necessary to encode polynucleotides of defined sequences that were much longer. Initially Khorana designed an amplification method for genes, seen on the left. This approach used synthetic oligonucleotides that are partially overlapping, with DNA polymerase used for extension. Next, the sequences were denatured or strands separated, and then hybridized with partly overlapping oligonucleotides. Next, the resulting products could be extended once again, and so on. The approach was similar to the polymerase chain reaction method; however, this approach was not necessary after the discovery of several new enzymes. Key discoveries included polynucleotide kinase, which could add a phosphate group to the 5'-end of oligonucleotides, required for sealing nicks with, DNA ligase, also a new enzyme. Next, restriction enzymes were discovered, which allowed DNA to be cut and pasted back, using the activity of DNA ligase.
	Slide #31: The first gene to be completely synthesized was yeast alanine tRNA which was 77 base pairs long. To accomplish this feat, the Khorana lab synthesized 17 oligonucleotides between 5 and 20 nucleotides long, which were designed to be in 3 overlapping segments. The complimentary ends were used to hybridize the three longer segments and the nicks between the segments were sealed with DNA ligase after phosphorylation with polynucleotide kinase. For the third segment, there were two alternative strategies, with one working better than the other, because of chemical properties of specific oligonucleotides. Khorana lab member, Marv Caruthers, also a pioneer in the synthesis of oligonucleotides, said that the synthesis of the first gene required an estimated 20 man-years of work.
	Slide #32: The next gene to be synthesized required even more time and effort. This was a functional tyrosine suppressor tRNA gene and was published in the year I joined Dr. Khorana's group. This gene was 126 base pairs long with 56 base pairs for the promoter and 25 for the 3’-terminator region. Khorana used 26 oligonucleotides of 16 nucleotide average length, which required considerably more synthesis. However, it was possible to show that this gene was functional in vivo by inserting it into either a plasmid or a phage vector and introducing it into E. coli. Because this was a suppressor tRNA, it could be shown to genetically suppress certain mutations in E. coli called amber mutations. When the lambda phage integrated into the chromosome, it could suppress either E. coli or lambda amber mutations. Also, accurate transcription and processing of the gene could be shown in vitro.
	Slide #33: After synthesis of two genes, Professor Khorana moved on to an entirely new field of research, membrane biology. The membrane protein that he focused on first was bacteriorhodopsin, which is produced by an unusual purple organism, called Halobacterium halobium. This microbe is primitive and a member of the third Domain of life, the Archaea. This protein was exceptional because it would form a two-dimensional lattice in a specialized part of the cell membrane, called the purple membrane. Electron microscopy was used to show that the purple membrane protein traverses the membrane seven times, with seven transmembrane alpha helical segments. Bacteriorhodopsin also binds to a retinal chromophore and catalyzes the transfer of hydrogen ions (or protons) across the membrane in response to light. Light drives protons from the inside to the outside of cells, creating a proton motive gradient which can be used by ATP synthase to drive ATP synthesis. With inside-out membrane vesicles containing bacteriorhodopsin and mitochondrial ATP synthase, light was shown to drive the synthesis of ATP, confirming the validity of the chemiosmotic coupling theory.
	Slide #34: Professor Khorana’s first major achievement with bacteriorhodopsin was to determine the sequence of amino acids in the protein. He wanted to understand how this protein catalyzes the transport of protons across the membrane, so he wanted to clone the bacteriorhodopsin gene and conduct structure-function analysis by mutagenesis. After sequencing the protein, he identified a four amino acid region which, based on the genetic code, had the least redundancy. A region near the N-terminus contained a four amino acid region with two tryptophans and a glutamic acid. The tryptophans are coded by a single codon, and glutamic acid is encoded by two codons, so this region was chosen for back translation, primers, and cDNA synthesis.
	Slide #35: Khorana’s laboratory made two oligonucleotides corresponding to both glutamic acid codons while guessing about the identity of the isoleucine codon, which is coded by three different codons in the genetic code. To make a long story short, Khorana's laboratory was able to use those primers to successfully synthesize a copy DNA or cDNA using the enzyme, reverse transcriptase, which makes DNA copies of RNA. The initial DNA product was about 80 nucleotides long, and because the 5’-end of the gene contains an inverted repeat, they also obtained the complementary second strand. Next, the double stranded cDNA could be cloned after treatment with single stranded nuclease to make blunt ends and addition of linkers. The cDNA was used as probe to clone the complete bacteriorhodopsin gene.
	Slide #36: The bacteriorhodopsin gene became one of the first archaeal genes to be cloned and sequenced, confirming that the Archaea also use the standard genetic code. In addition, the bacteriorhodopsin gene sequence showed the protein contains a very unusual signal sequence, about 13 amino acids long, at the 5’ end. The sequence upstream of the gene also showed that the bacteriorhodopsin gene did not have a typical bacterial type promoter for transcription initiation. One of my projects in the Khorana lab was to determine whether or not a promoter was indeed present immediately upstream of the gene or whether the gene was part of an operon with the promoter located much further upstream.
	Slide #37: The way in which I proceeded in this project was by purification of the messenger RNA and 5’-end analysis. The mRNA was purified by a method called hybrid selection and tested for labeling at the 5’-end using a viral capping enzyme. This was a eukaryotic RNA processing enzyme which requires a GTP. Since primary transcripts have a triphosphate at the 5’-end and can be capped in vitro using the capping enzyme, if the alpha phosphate is labeled, the red one, then the label ends up at the 5’-end of the messenger RNA. When the labeled messenger RNA was hydrolyzed using nuclease enzymes, the base at the 5’-end could be identified. In the left panel is seen that the 5'-end was a G nucleotide. Next, by conducting RNA sequencing using a method called two-dimensional homochromatography, the sequence of the 5'-end of the RNA could be determined to be GC-AUG-UUG. This showed the bacteriorhodopsin gene mRNA contained a two nucleotide leader, an AUG methionine start and a UUG leucine second codon. These results showed for the first time that a novel promoter was indeed present immediately upstream of the bacteriorhodopsin gene and the archaeal transcription system was novel.
	Slide #38: Another project that I worked on in Dr Khorana's lab was the development of Halobacterium genetics. In particular, I studied bacteriorhodopsin mutants and discovered transposable elements which inactivate the bacteriorhodopsin gene. Mutants of the Halobacterium bacteriorhodopsin overproducer were observed at high frequency and there were of two different types – white and orange. I focused on the orange variety, which had lost the ability to produce only the purple membrane, as seen in the sucrose gradients at the bottom. On the right is a genetic restriction map showing where the insertions occurred within or around the hatched bacteriorhodopsin gene. Two different types of insertion elements were discovered, called ISH1 and ISH2, for insertion sequence 1 and 2 from Halobacterium.
	Slide #39: The ISH1 elements inserted into the bacteriorhodopsin gene at a specific site in both orientations, and this could be seen both from DNA sequencing analysis and DNA imaging in the electron microscope, using a technique called heteroduplex mapping. The ISH2 element could insert at different locations in the gene, including a site that was a hundred bases upstream of the transcription start point. This mutation helped us to define the archaeal promoter. This work was instrumental in the pre-genomics era to establish the value of genetics of diverse microorganisms. It also led to the sequencing of the Halobacterium genome many years later.
	Slide #40: The research I did in Dr. Khorana's lab helped to launch my independent career in archaeal biology, which I continued after post-doctoral training at Harvard Medical School, Massachusetts General Hospital, and a visiting position at the Pasteur Institute. First, I was a faculty member at the University of Massachusetts in Amherst and later at the University of Maryland in Baltimore. The journal and book covers shown reflect some of the important discoveries made possible by the superb graduate education I received in the Khorana laboratory.
	Slide #41: Dr. Khorana, however, kept his laboratory's focus on his original goal, one of his main scientific characteristics. His goal was to determine the mechanism of light-driven transmembrane ion translocation. He accomplished this goal by proceeding to synthesize the bacteriorhodopsin gene, which was 757 base pairs long. It required 28 synthetic oligonucleotides ranging in size from 21 to 69 nucleotides. Dr. Khorana added an E. coli promoter and expressed the bacteriorhodopsin gene in this bacterial host. He reconstituted the functional protein produced in E. coli by addition of retinal and insertion into membrane lipid vesicles. His lab also did an enormous amount of site-directed mutagenesis and carried out extensive functional analysis.
	Slide #42: This was a tremendous amount of work and resulted in some seminal findings. For example, he found that retinal binds to lysine 212 as a Schiff base and that the proton in the Schiff base is pumped out of the cell during bacteriorhodopsin photocycling that occurs when illuminated with light. Ion translocation across the membrane occurs by first transfer of the Schiff base proton to aspartic acid 85 shown in red prior to extrusion to the outside. That event is followed by uptake of a proton from the inside to aspartic acid 96 and then transfer of the proton to the retinal-lysine 212 Schiff base. That completes the photocycle, resulting in pumping of a proton from the inside to the outside of the cell in response to light.
	Slide #43: While working toward his intended goal of studying bacteriorhodopsin ion translocation, Professor Khorana initiated studies on another membrane protein, rhodopsin which is part of the mammalian visual system. Rhodopsin, while not being an ion pump, has a bound retinal chromophore. In this case it is a 11-cis isomer rather than an all-trans isomer as in bacteriorhodopsin. Both bind to a lysine in the seventh transmembrane alpha helix. Rhodopsin is also a member of the seven transmembrane protein family, called GPCRs, and is a light receptor that performs a vital function in vision. However, rhodopsin absorbs light and interacts with a G protein, transducin, as shown in the textbook diagram. Transducin has three subunits and initiates a cascade of events responsible for signaling in mammalian vision. In this work, the Khorana lab also proceeded to synthesize the rhodopsin gene, which is even larger than the bacteriorhodopsin gene,1,057 base pairs long. Synthesis required 72 oligonucleotides between 15 and 40 in size, and a similar strategy was used for complete synthesis.
	Slide #44: Dr. Khorana's laboratory spent about 20 years studying rhodopsin structure and function and signal transduction, and there were many remarkable achievements - too many achievements to detail here. To summarize a few, after gene synthesis rhodopsin protein was expressed in mammalian cells. After purifying and reconstituting rhodopsin, his lab also synthesized and expressed two subunits of the G-protein transducin. The methods that his laboratory developed are widely used for studying these types of membrane proteins, which are very common in mammalian cells, and are drug targets. He identified the G-protein (transducin) binding site on rhodopsin, he also identified the retinal binding pocket and the Schiff base counter ion that is important for spectral tuning. Khorana's lab also identified the first rhodopsin mutant responsible for autosomal retinitis pigmentosa, which causes blindness. Those are some of the highlights of major research accomplishments of Professor Khorana's laboratory over the course of six decades.
	Slide #45: In the final part of the talk, I would like to turn to Professor Khorana's inspiration and legacy. Dr. Khorana was an inspiring mentor as he set an example to his students and others. He was highly committed to free and open scientific pursuit and he was completely dedicated to teaching the most rigorous scientific method to his students and postdoctoral associates. And he had an unending need to expand our knowledge through scientific research. Why did he do this? Well, this quote indicate the importance of science to Dr. Khorana: “Such scientific work would be necessary to solve the environmental, economic, and health problems that face humanity. I do have the basic faith that the survival of our civilization is not even going to be possible without the proper use of science."
	Slide #46: Professor Khorana's scientific legacy is nothing less than monumental. His accomplishments established the foundations of modern chemistry and biology. The genetic code work, the code itself, is routinely used to translate nearly all sequenced genomes into protein products. And thousands of genomes have been sequenced with almost all using the standard code, with very few exceptions. Oligonucleotide synthesis, which is now routine and automated, is used for gene synthesis and expression, mutagenesis, and recently even whole genome synthesis. This is a new field called synthetic biology. The many techniques developed for working on membrane proteins, from pioneering studies of bacteriorhodopsin and rhodopsin a,re commonly employed for many other membrane proteins, including many drug targets. And Professor Khorana's six decade-long career resulted in training of hundreds of postdoctoral associates, and a small number of graduate students. All have been inspired by Professor Khorana's commitment to science and legendary achievements and they carry the mantle of rigorous science that he established.
	Slide #47: I want to conclude with just a few more quotes from Professor Khorana, including this early one highlighting his vision. He said, after the genetic code was deciphered: “I wish to conclude by hazarding the following rather long range predictions. In the years ahead, genes are going to be synthesized. The next steps would be to learn to manipulate the information content of genes and to learn to insert them into and delete them from the genetic systems. When, in the distant future, all this comes to pass, the temptation to change our biology will be very strong.”
	Slide #48: Professor Khorana was also very generous, both to his students and his teachers. About his teachers, he said: “In my own scientific development, I was most fortunate in coming under the influence of a number of very great scientists: Vladimir Prelog made me see the beauty in chemistry, work and effort. Later, in biochemistry, I came under the influence of Fritz Lipmann, who was so gifted in integrating ideas, and Arthur Kornberg, who taught me stringency in biochemical experimentation. Association with Francis Crick during and since work on genetic code has been intellectually stimulating and inspiring. Much later, Efraim Racker introduced me to membrane biochemistry.”
	Slide #49: I had the pleasure of visiting Professor Khorana at MIT after his move to a new biology building in 1995, when he invited me to give a seminar to his group. On that occasion, I presented him with a copy of Rabindranath Tagore's Gitanjali, and afterward, he wrote me this letter: “Dear Shil, I would fondly treasure the 1913 edition of Gitanjali that you brought me. Thank you deeply for this unique gift. It also brings back many memories of my youthful days when we held Rabindranath Tagore in great affection and respect for his spiritual leadership in our Independence movement. Best wishes, Gobind”.
	Slide #50: In closing, Dr Khorana was a visionary and he was inspirational. He made contributions to science which will last another hundred years, and perhaps longer… The world owes this great man a debt of gratitude. Thank you.
Thanks are due to the Nobel Foundation, University of British Columbia, University of Wisconsin, MIT, and the Royal Society for assistance and permission for resources used in this presentation. ©Shiladitya DasSarma