Computer Programmers Have It Easy

Computer programmers can write a piece of code in just a few minutes before running it and seeing the results. Biologists manipulate code of a different kind, spending days modifying gene sequences. However, they plod along as they clone plasmids—the loops of DNA that biologists use to manipulate and study organisms—because propagating them relies, in part, on the pace at which cells grow and divide. Due to the nearly ubiquitous use of cloning in life science research, this lost time adds up.

Researchers use DNA cloning to make numerous copies of a gene, which can then be inserted into living cells to study how that gene functions or otherwise coax the cell to make proteins. Most medicines, including insulin and semaglutide (the weight loss drug), are made using DNA cloning. The ability to clone DNA faster would enable scientists to run experiments, make discoveries, and produce drugs more quickly.

During my doctoral training, I split my work between wet lab research and programming, two streams that differed radically in terms of productivity. The days I spent coding gratified me: I could write several functions in a single sitting and see the fruits of my labor in no time at all. Conversely, the days I spent modifying genes frustrated me because it often took weeks before I could transfect my modified plasmids, carrying genes for fluorescent proteins, into human cells.

The cloning process itself contributed to this delay, requiring multiple steps, such as cutting and pasting sequences from one plasmid into another, excising antibiotic resistance genes, or flipping sequences around. For each perturbation, researchers like me must re-clone the modified DNA sequences into bacteria, such as Escherichia coli, to mass produce the plasmid. However, E. coli grows slowly, doubling only every 20 minutes under the best conditions, forcing us to wait overnight for bacterial colonies to appear on culture plates. We must then scale up colonies into large populations by culturing them in liquid growth media, adding another 24 hours, after which we can finally isolate the DNA. Thus, each round of cloning takes at least three days of work.

And that’s only if everything goes well. Sometimes cells don’t take up the DNA, or unforeseen issues with the modified sequence arise—such as off-target mutations, unexpected protein modifications, or disruptions to gene regulation—that send us back to the drawing board. These setbacks can extend the timeline from days to weeks. In contrast, coding errors, while expected, disrupt productivity far less—so much so that programmers joke that “code never works the first time around.” Programmers can afford to hurriedly craft code and make small mistakes like misplacing commas because, unlike biologists grieving over their failed DNA constructs and delayed experiments, they can correct errors in seconds.

Biophysical Limits

In recent years, researchers have found biophysical limits on cell division speeds, such as genome size or the time-consuming synthesis of bulky ribosomes, the large cellular complexes that build proteins. Even so, several avenues exist to make cells divide faster, such as by simplifying genomes and ribosomes or enlisting artificial selection to favor genetic mutations that boost speed. Insofar as virtually every field in biological research uses E. coli to clone DNA, engineering this microbe to divide more quickly would greatly accelerate research.

Comparing the growth speed of different organisms might steer us toward ways of jump-starting this process. The rate at which organisms double is central to how evolutionary biologists weigh up the competitiveness of individuals within a species. Evolutionary fitness boils down to two factors: survival and—of greater relevance here—reproduction. I’ve always admired how these two factors underpin all the world’s diversity. The fittest individuals will outnumber and outlive their slower-growing kin, producing large populations of cells that pass genes conferring adaptations down generations.

If we compare all lifeforms by their speed of cell division, multicellular eukaryotic cells reproduce the slowest. So-called primary cells, harvested from living tissues and cultured in the laboratory, can take weeks to grow and spread across a culture flask, hampered partly by their large genomes, which take time to copy. It takes about eight hours for the fastest-dividing human cells to double their DNA. In addition, they carry out two intermediate steps between rounds of replication and division, known as G1 and G2 phase, stalling division by roughly 15 more hours. Primary cells also begin to divide more slowly or come to a halt after a few rounds of doubling, making them inconvenient for long-term experiments. For this reason, researchers often use rapidly proliferating cancer cells, such as HeLa cells, which double every 17 hours indefinitely.

Some bacteria, like Mycobacterium tuberculosis, reproduce as slowly as eukaryotes, taking 24 hours to double under the best conditions. However, most bacteria are built for speed. Among the fastest, E. coli and Bacillus subtilis take a mere 20 minutes to divide. Vibrio natriegens divides faster still, doubling in only ten minutes. Bacteria benefit from being single-celled,¹ having tiny genomes hundreds of times smaller than those of eukaryotes, and lacking an intermission between rounds of replication and division. So, when looking to design swift cells that can elevate productivity, these microbes make great contenders.²

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What’s the Delay, Then?

“A bacterium's goal is to become bacteria,” said the late microbiologist, Stanley Falkow. Watch a timelapse of bacteria doubling and you get the impression they don’t do much else. E. coli exemplifies Falkow’s dictum; though it sounds impossible, these bacteria copy their genome every 40 minutes yet divide in half that time. They achieve this feat by starting new rounds of replication even before previous ones have finished, juggling up to a dozen rounds at any given time. Unlike eukaryotic cells that pause replication for the majority of the cell cycle, these multitasking bacteria never take a break.

By marveling at fast-lane cruisers like E. coli, we witness how evolution favors quickly dividing cells. Yet, despite refinements over 3.5 billion years, hindrances creep in and limit natural selection. In the same way that a race car’s tires wear away at fast speeds, cells struggle to indefinitely increase their velocity without having it take a toll on their health. They also can’t come up with new strategies from scratch the way an engineer might; they must “retrofit” existing genes to adapt, making it difficult to circumvent bottlenecks.

Genome size creates the first bottleneck to cell division. Larger genomes generally take longer to copy because as a DNA polymerase enzyme runs along a DNA strand during replication, it pauses to proofread the copied sequences. Evolution hits the brakes to avoid burning its tires: If polymerases rushed this phase, the cell might accrue too many harmful mutations. Although the rates vary, a prokaryotic polymerase copies 1,000 bases per second on average, but a eukaryotic one copies only 30 in the same time. Though slower, eukaryotic polymerases make up for this by initiating replication at multiple sites along chromosomes, thus dividing the job between multiple enzymes. Bacteria don’t split up this task in the same way. They begin replication at just one locus, with two polymerases running in opposite directions to copy the whole genome.

Eukaryotic polymerases move slowly because they check each base added at the polymerase active site, and if a mismatch is detected, the newly-formed DNA strand is shunted to a distant region of the polymerase complex that harbors an exonuclease active site to cleave off the erroneous base.

The proofreading step for each new base requires large conformational changes in the DNA polymerase that biophysicists at the University of Oxford suspect limit the rate of the enzyme. The polymerase resembles a hand with fingers and a thumb, and as it proofreads a replicating strand it repeatedly opens and closes the gap between its thumb and fingers. To see how quickly the polymerase shifts between “open” and “closed” states, the researchers labeled the fingers and thumb with different fluorescent dyes. The one on the fingers shines by default, whereas the one on the thumb remains in an “off” state. When they come together, a process called Förster resonance energy transfer takes place: The fingers’ dye donates its fluorescence to the thumb’s dye, switching it “on.” When the polymerase “hand” opens again, the dyes revert back to their original states. By tracing how frequently the thumb’s dye flashed, the researchers determined that opening is five times slower, and likely impedes the rate of DNA replication.

The rate at which polymerase enzymes fulfill their function illustrates that genome size acts as a fundamental barrier to speedy replication. Smaller genomes, therefore, could lead to faster-growing cells.

The biosynthesis of ribosomes, the complexes that build all the cell’s proteins, causes an additional bottleneck. Ribosomes create all the enzymes needed to catalyze chemical reactions important for growth, and ramping up enzyme production could, in theory, speed up cell division times. However, this would require the cell to make more ribosomes which, in turn, would slow down division speeds because the production of these Behemoth complexes requires copious resources and time.

For a 2021 paper, researchers at the California Institute of Technology estimated how long it would take E. coli to churn out a single ribosome using back-of-the-envelope calculations. RNA and proteins comprise each ribosome, but the cell synthesizes these two biomolecules at different rates. The RNA polymerase enzyme transcribes DNA into RNA at a rate of approximately 40 bases per second, but proteins emerge much more slowly: only around 15 amino acids are added to a growing protein chain in the same amount of time. With approximately 50 proteins in a ribosome, totaling approximately 7,500 amino acids, they estimated that ribosomal protein synthesis is one of the slowest processes in E. coli. It takes approximately eight minutes for a cell to synthesize the proteins needed to make just one ribosome. In that time, we could listen to ‘The Speed of Life’ by David Bowie—three times!

Suppose E. coli possessed only one ribosome, how long would it take the cell to divide? If this one ribosome added 15 amino acids to growing protein chains every second, and if the total protein content of the cell amounts to one billion linked amino acids, it would take over two years to build up all the cell’s proteins.

An E. coli divides every 20 minutes, then, because it spreads its molecular labor across anywhere from 10,000 to 100,000 ribosomes. These estimates match experimentally determined ribosome concentrations, suggesting that ribosomes already work at maximum capacity in these microbes.

When ribosomal proteins evolved, they became integral to cell survival. But as cellular life sped up, synthesizing these proteins became a time-consuming burden that cells couldn’t abandon without losing other crucial functions.

Ribosome synthesis may take the most credit for limiting division rates, but the cell has yet other bottlenecks to contend with. In 2011, biophysicists at Stony Brook University estimated how the rates of protein folding impact E. coli growth. They found that some proteins fold much slower than others, depending on their three-dimensional structures. Helical twists in a protein form faster than parallel-running strands called β-sheets, for example. Some proteins take as much time to fold as an entire E. coli cell takes to divide. This suggests that even if cells could sidetrack the time-consuming synthesis of ribosomes, slow protein folding could be another roadblock to faster division.

Making Cells Go Faster

Decluttering the genome might make cell division faster; remove genes and leave behind only the bare essentials. But this approach would only work if researchers take extreme care to avoid deleting genes that actually facilitate growth.

Scientists are currently fashioning two minimalist microbes. A research team in California has purged the genome of Mycoplasma mycoides capri, one of the smallest known bacteria, to create a minimal cell called JCVI-syn3A, half its original size. A different team in Germany plans to minimize Bacillus subtilis, creating a MiniBacillus system, by clearing out a whopping 85 percent of its genes.

However, scrapping non-essential genes poses difficulties of its own. Researchers typically identify “surplus” genes by deleting them one by one and verifying that the growth and survival of the cells don’t change. But some genes compensate for each other, leading to detrimental effects in their combined absence. For example, E. coli’s genome encodes two enzymes to make the amino acid asparagine. Remove either one of the genes and the bacteria cope fine. Remove both, however, and they die. Without at least one of the genes, the bacteria can no longer synthesize this essential building block.

Scientists must take care not to accidentally delete genes that influence growth, a counterproductive excision that can drastically slow down division speeds. For example, the more researchers cut back on the JCVI-syn3A genome, the longer the cell takes to divide. The third and most recent version of this minimal Mycoplasma doubles up in twice the time as its predecessors. Although the MiniBacillus cell hasn’t been created yet, the team behind the project predicts it will have a doubling time of less than one hour—far longer than the 20-minute division time of the original bacterium.

These case studies warn that minimizing genomes doesn’t necessarily improve growth rates, even though it should be theoretically possible.

Tinkering with origins of replication, sequences on the bacterial chromosome that kick off replication, might also encourage cell division. DnaA, a protein that initiates replication, recognizes sequences at the origin and then splits apart the two DNA strands. Other replicative enzymes then dock onto the exposed strands and begin copying them.

E. coli has one origin, but synthetic biologists at Yonsei University in South Korea found that adding two extra origins at different sites on the chromosome could propel cell division. They worked with a slow-dividing E. coli strain called MG1655, which normally takes one hour to double up. By adding two more origins, they shaved 4 minutes off the doubling time. Though they only achieved a small boost, the data is encouraging: the cells were viable despite carrying extra origins, and the findings reveal that tweaking the number and position of these replication start sites could speed up cell division rates. It would be interesting to see if adding surplus origins in fast-dividing E. coli strains could achieve a similar effect.

Another approach could involve moving genes around. Scientists recently found that replication influences the order in which genes on the bacterial chromosome are expressed. During a replication cycle in E. coli, genes are switched on in the order they are copied, starting with genes located near the origin, thus producing extra copies of those genes sooner and boosting their expression. Researchers could consider shuffling genes around so that they would position those that accelerate growth near the origin.

For a 2021 study, researchers used minimal Mycoplasma cells to pinpoint genes essential for cell division, finding seven. The most notable genes include ftsZ and sepF, which encode proteins that split a dividing cell into two by assembling into a constricting ring that tightens and splits apart the cell membrane at the dividing line between budding cells. Without this cell-splitting machinery, Mycoplasma cells inflate and swell—sometimes to 25 times their typical volume—but do not divide. One could speculate that placing these cell-splitting genes near the origin might ensure they are always present at an adequate concentration to split cells and thus speed up cell division. However, tuning cell-splitting enzymes to the right levels could be a tricky balancing act. Overexpressing these genes might cause cells to divide too early before all the essential components have duplicated. Nonetheless, they are good candidates for genes to be relocated in order to speed up cell division.

Additionally, rather than trimming genomes, perhaps doubling times could be reduced by trimming down ribosomes. Recall that a typical ribosome in bacteria is built from 50 proteins and 3 strands of RNA. It is the RNA that catalyzes chemical reactions: the fusions of amino acids into a variety of polypeptide chains that fold into proteins. Structural biologists have already designed blueprints for a minimalistic yet functional ribosome. Leaner ribosomes might take less time to synthesize, and therefore boost speeds.

To make the blueprint, scientists at the University of Alabama at Birmingham figured out which parts of the ribosome are essential for its function and should therefore be left untouched. Using structures of the whole complex resolved by cryo-electron microscopy and X-ray crystallography, they highlighted ribosome residues conserved across bacteria, archaea, and eukaryotes. Conserved regions huddled near the core of the ribosome, suggesting ones at the periphery may be more dispensable. However, no one has tried to synthesize and test out the functionality of a minimal ribosome by removing these potentially superfluous parts.

The researchers identified 27 ribosomal proteins not conserved across bacteria, archaea, and eukaryotes. How much time could the cell save in synthesizing ribosomes if it relinquished these components? To match the earlier estimates made by researchers at Caltech, if you assume the ribosome is made up of 50 proteins of equal size, totaling 7500 amino acids, then these 27 would account for roughly 4050 amino acids. At a rate of 15 amino acids added to the growing protein per second, researchers could theoretically shave off about four minutes—half the time taken to synthesize a ribosome—by omitting these proteins. In practice, some of these proteins may prove crucial for E. coli ribosomes to function, even if they’re not conserved across the three domains of life, so reducing the ribosome to this scale might not work.

Encouraging what nature has already created offers another option; researchers could use artificial selection to increase growth rates. The carefully curated conditions in a laboratory create a perfect setting to investigate mutations that boost proliferation and promote artificial selection. Out in nature, selection doesn’t always favor high-speed reproduction. Cells must contend with changing environments and new stressors, and mutations that enhance survival (the other half of the fitness equation) may prevail in these circumstances, rather than ones that favor reproduction. In contrast, by setting up optimal, constant growth conditions in the laboratory, cells may face fewer survival pressures and adopt adaptations for briskness instead. As novel speed adaptations accumulate in laboratory cultures, researchers can cherry-pick bacteria that divide faster.

Evolutionary biologists at University Bourgogne Franche-Comté in France have found success by artificially selecting microbial communities carrying up to 16 different bacterial strains. Their research revealed that, after 40 cycles of growth and selective propagation, the most diverse communities grew into the largest populations, potentially thanks to rapid division. They speculated that having a greater amount of variation at the start creates more opportunities for genes to interact synergistically and promote proliferation.

Going Offroad

Nature contains a tremendous amount of variation, and so discovery and not design may equip biologists with faster-dividing bacteria. 66 years ago, when researchers on Sapelo Island, Georgia took off their lab coats and put on their Wellington boots, they stumbled upon a bacterial speed demon in salt marshes, called Vibrio natriegens. This microbe’s hasty division has recently drawn the attention of scientists. In fact, with a minimum doubling time of 9.4 minutes (twice the speed of E. coli ), it holds the place as one of the fastest-dividing organisms known to science, making this species a potential game-changer for biotechnology.

Colonies of V. natriegens appear on agar plates in as little as six hours as opposed to at least 14 hours for E. coli. Only Clostridium perfringens divides faster, doubling approximately every six minutes. However, Clostridium perfringensproves tricky to work with in the lab. For one thing, being strictly anaerobic, it doesn’t survive well when exposed to oxygen. Plus, as a human pathogen that causes gut infections, it requires extra precautions while handling it.

V. natriegens, on the other hand, could make a great laboratory model. It is non-pathogenic plus easy to culture and genetically modify. Synthetic biologists at Synthetic Genomics, Inc. in California have already minimized its genome for industrial purposes, excluding 194,000 DNA bases. Rather than deleting genes one by one to test their importance—as was done to shrink JCVI-Syn3A and MiniBacillus—the researchers compared the chromosomes of two V. natriegensstrains side by side. This allowed them to spot differences in their genomes, highlighting regions that weren’t conserved and could be jettisoned. The researchers chose to discard genes that encode virus-sensing machinery because the cells would be grown in a sterile laboratory environment. They also removed a gene that could hamper efforts to clone DNA into the microbe; it encoded an extracellular nuclease that digests DNA in the bacterium’s environment, including plasmids intended for cloning. The resulting strain of V. natriegens proves simpler to engineer than its wild counterpart.

Ultimately, no matter the model organism or where it comes from, molecular biology will probably never approach the speed of computer programming. But it can improve. Imagine a future where E. coli or V. natriegens divide twice as fast. No longer would researchers have to put their experiments on hold for days while waiting for bacterial colonies to form on Petri dishes. Instead, they could complete multiple cloning steps per day, waiting only a few hours for bacterial colonies to appear. In the same vein that errors in computer code can be quickly corrected once written, cloned DNA could be swiftly scrutinized for faults, leading a scientist to repeat merely a week’s worth of work if needed. Perhaps future biologists will experience the same freedom of improvisation as programmers drafting code. They might even wink at their own running joke that “cloning never works the first time.”

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Kamal Nahas is a researcher-turned-journalist based in Oxford, UK, who covers stories in biology, health, and technology.

Cite this essay: Kamal Nahas. “Breaking the Speed Limit on Cell Division.” Asimov Press (2024). DOI: https://doi.org/10.62211/23ty-56hw

Thanks to Merrick Pierson Smela and Tom Ellis for reading drafts of this essay.

Editor’s Note: Niko McCarty, an editor of this essay, worked in Rob Phillips’ group at Caltech, which estimated the speed of ribosome synthesis.

Footnotes

1. Some bacteria form multicellular communes, even with other bacterial species, that limit and synchronize cell division to prevent one “cheater” bacterium from dividing at the expense of the whole. Also called consortia, these bacteria have useful applications in industry. By secreting and sharing resources, they can produce molecules beyond the scope of individual species.
2. Prokaryotes are the fastest dividers among cellular lifeforms, but I would be remiss if I didn’t give a nod to viruses, bacteriophages (or bacterial viruses), and mobile genetic elements (or jumping genes). These lifeforms parasitize cells and can hijack fast-dividers for their own replication, often producing multiple progeny and exceeding their hosts at proliferation. Within an hour of a T4 bacteriophage infecting an E. coli bacterium, hundreds of viral offspring burst out of the cell.

This article was published on July 14, 2024.