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Some time last year, I found an article on Wikipedia about the smallest something to be able to reproduce.
I don't remember exactly what it was, but I am fairly certain that after the initial discovery another of the previous organism (this one slightly smaller) was discovered.
I think that the smallest something might have been the smallest self-replicating protein, or smallest self-replicating molecule, or something like that.
It was not mentioned in this thread: Which organism has the smallest genome length?
It had a strange, stand-out name and I believe it was discovered in the 90s.
You're probably thinking of the Spiegelman Monster. It was actually discovered in 1965, but it was discovered that it became shorter over time in 1997.
It also wasn't included in that thread, and it has a strange name.
A research team (Lee et al.) has discovered what maybe the smallest known self replicating RNA molecule - actually a 32-unit-long "a-helical peptide" whatever that is. It is based on the yeast transcription factor GCN4. The 32 peptide sequence is so small, that one could describe it in text form simply as:
ArCONH--RMKQLEEKVYELLSKVA-CLEYEVARLKKLVGE--CONH2 ArCONH--RMKQLEEKVYELLSKVA-COSBn H2N--CLEYEVARLKKLVGE--CONH2
Two of those lines are likely to be pre-cursor molecules required (the food), and one is likely to be the RNA sequence, I think the final line.
This is a lot smaller than the smallest known or simplest genome, which could be Carsonella ruddii, which lives off sap-feeding insects, has taken the record for smallest genome with just 159,662 'letters' (or base pairs) of DNA and 182 protein-coding genes. Source: https://www.nature.com/news/2006/061009/full/news061009-10.html
Also, it's likely you should checkout this study: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187685/ "Systems Biology Perspectives on Minimal and Simpler Cells "
First Self-Replicating, Synthetic Bacterial Cell Constructed by J. Craig Venter Institute Researchers
ROCKVILLE, MD and San Diego, CA (May 20, 2010) — Researchers at the J. Craig Venter Institute (JCVI), a not-for-profit genomic research organization, published results today describing the successful construction of the first self-replicating, synthetic bacterial cell. The team synthesized the 1.08 million base pair chromosome of a modified Mycoplasma mycoides genome. The synthetic cell is called Mycoplasma mycoides JCVI-syn1.0 and is the proof of principle that genomes can be designed in the computer, chemically made in the laboratory and transplanted into a recipient cell to produce a new self-replicating cell controlled only by the synthetic genome.
This research will be published by Daniel Gibson et al in the May 20 th edition of Science Express and will appear in an upcoming print issue of Science.
"For nearly 15 years Ham Smith, Clyde Hutchison and the rest of our team have been working toward this publication today--the successful completion of our work to construct a bacterial cell that is fully controlled by a synthetic genome," said J. Craig Venter, Ph.D., founder and president, JCVI and senior author on the paper. "We have been consumed by this research, but we have also been equally focused on addressing the societal implications of what we believe will be one of the most powerful technologies and industrial drivers for societal good. We look forward to continued review and dialogue about the important applications of this work to ensure that it is used for the benefit of all."
According to Dr. Smith, "With this first synthetic bacterial cell and the new tools and technologies we developed to successfully complete this project, we now have the means to dissect the genetic instruction set of a bacterial cell to see and understand how it really works."
To complete this final stage in the nearly 15 year process to construct and boot up a synthetic cell, JCVI scientists began with the accurate, digitized genome of the bacterium, M. mycoides. The team designed 1,078 specific cassettes of DNA that were 1,080 base pairs long. These cassettes were designed so that the ends of each DNA cassette overlapped each of its neighbors by 80bp. The cassettes were made according to JCVI's specifications by the DNA synthesis company, Blue Heron Biotechnology.
The JCVI team employed a three stage process using their previously described yeast assembly system to build the genome using the 1,078 cassettes. The first stage involved taking 10 cassettes of DNA at a time to build 110, 10,000 bp segments. In the second stage, these 10,000 bp segments are taken 10 at a time to produce eleven, 100,000 bp segments. In the final step, all 11, 100 kb segments were assembled into the complete synthetic genome in yeast cells and grown as a yeast artificial chromosome.
The complete synthetic M. mycoides genome was isolated from the yeast cell and transplanted into Mycoplasma capricolum recipient cells that have had the genes for its restriction enzyme removed. The synthetic genome DNA was transcribed into messenger RNA, which in turn was translated into new proteins. The M. capricolum genome was either destroyed by M. mycoides restriction enzymes or was lost during cell replication. After two days viable M. mycoides cells, which contained only synthetic DNA, were clearly visible on petri dishes containing bacterial growth medium.
The initial synthesis of the synthetic genome did not result in any viable cells so the JCVI team developed an error correction method to test that each cassette they constructed was biologically functional. They did this by using a combination of 100 kb natural and synthetic segments of DNA to produce semi-synthetic genomes. This approach allowed for the testing of each synthetic segment in combination with 10 natural segments for their capacity to be transplanted and form new cells. Ten out of 11 synthetic fragments resulted in viable cells therefore the team narrowed the issue down to a single 100 kb cassette. DNA sequencing revealed that a single base pair deletion in an essential gene was responsible for the unsuccessful transplants. Once this one base pair error was corrected, the first viable synthetic cell was produced.
Dr. Gibson stated, "To produce a synthetic cell, our group had to learn how to sequence, synthesize, and transplant genomes. Many hurdles had to be overcome, but we are now able to combine all of these steps to produce synthetic cells in the laboratory." He added, "We can now begin working on our ultimate objective of synthesizing a minimal cell containing only the genes necessary to sustain life in its simplest form. This will help us better understand how cells work."
This publication represents the construction of the largest synthetic molecule of a defined structure the genome is almost double the size of the previous Mycoplasma genitalium synthesis. With this successful proof of principle, the group will now work on creating a minimal genome, which has been a goal since 1995. They will do this by whittling away at the synthetic genome and repeating transplantation experiments until no more genes can be disrupted and the genome is as small as possible. This minimal cell will be a platform for analyzing the function of every essential gene in a cell.
According to Dr. Hutchison, "To me the most remarkable thing about our synthetic cell is that its genome was designed in the computer and brought to life through chemical synthesis, without using any pieces of natural DNA. This involved developing many new and useful methods along the way. We have assembled an amazing group of scientists that have made this possible."
As in the team's 2008 publication in which they described the successful synthesis of the M. genitalium genome, they designed and inserted into the genome what they called watermarks. These are specifically designed segments of DNA that use the "alphabet" of genes and proteins that enable the researcher to spell out words and phrases. The watermarks are an essential means to prove that the genome is synthetic and not native, and to identify the laboratory of origin. Encoded in the watermarks is a new DNA code for writing words, sentences and numbers. In addition to the new code there is a web address to send emails to if you can successfully decode the new code, the names of 46 authors and other key contributors and three quotations: "TO LIVE, TO ERR, TO FALL, TO TRIUMPH, TO RECREATE LIFE OUT OF LIFE." - JAMES JOYCE "SEE THINGS NOT AS THEY ARE, BUT AS THEY MIGHT BE."-A quote from the book, "American Prometheus" "WHAT I CANNOT BUILD, I CANNOT UNDERSTAND." - RICHARD FEYNMAN.
The JCVI scientists envision that the knowledge gained by constructing this first self-replicating synthetic cell, coupled with decreasing costs for DNA synthesis, will give rise to wider use of this powerful technology. This will undoubtedly lead to the development of many important applications and products including biofuels, vaccines, pharmaceuticals, clean water and food products. The group continues to drive and support ethical discussion and review to ensure a positive outcome for society.
Funding for this research came from Synthetic Genomics Inc., a company co-founded by Drs. Venter and Smith.
The research published today was made possible by previous breakthroughs at JCVI. In 2007 the team published results from the transplantation of the native M. mycoides genome into the M. capricolum cell which resulted in the M. capricolum cell being transformed into M. mycoides. This work established the notion that DNA is the software of life and that DNA dictates the cell phenotype.
In 2008 the same team reported on the construction of the first synthetic bacterial genome by assembling DNA fragments made from the four chemicals of life — ACGT. The final assembly of DNA fragments into the whole genome was performed in yeast by making use of the yeast genetic systems. However, when the team attempted to transplant the synthetic bacterial genome out of yeast and into a recipient bacterial cell, viable transplants could not be recovered.
Ethical Considerations: Since the beginning of the quest to understand and build a synthetic genome, Dr. Venter and his team have been concerned with the societal issues surrounding the work. In 1995 while the team was doing the research on the minimal genome, the work underwent significant ethical review by a panel of experts at the University of Pennsylvania (Cho et al, Science December 1999:Vol. 286. no. 5447, pp. 2087 — 2090). The bioethical group's independent deliberations, published at the same time as the scientific minimal genome research, resulted in a unanimous decision that there were no strong ethical reasons why the work should not continue as long as the scientists involved continued to engage public discussion.
Dr. Venter and the team at JCVI continue to work with bioethicists, outside policy groups, legislative members and staff, and the public to encourage discussion and understanding about the societal implications of their work and the field of synthetic genomics generally. As such, the JCVI's policy team, along with the Center for Strategic & International Studies (CSIS), and the Massachusetts Institute of Technology (MIT), were funded by a grant from the Alfred P. Sloan Foundation for a 20-month study that explored the risks and benefits of this emerging technology, as well as possible safeguards to prevent abuse, including bioterrorism. After several workshops and public sessions the group published a report in October 2007 outlining options for the field and its researchers.
Most recently in December of 2008, JCVI received funding from the Alfred P. Sloan Foundation to examine ethical and societal concerns that are associated with the developing science of synthetic genomics. The ongoing research is intended to inform the scientific community as well as educate our policymakers and journalists so that they may engage in informed discussions on the topic.
For many biologists the clincher came in 2000, when the structure of the protein-making factories in cells was worked out. This work confirmed that nestling at the heart of these factories is an RNA enzyme – and if proteins are made by RNA, surely RNA must have come first.
Still, some issues remained. For one thing, it remained unclear whether RNA really was capable of replicating itself. Nowadays, DNA and RNA need the help of many proteins to copy themselves. If there ever was a self-replicator, it has long since disappeared. So biochemists set out to make one, taking random RNAs and evolving them for many generations to see what they came up with.
By 2001, this process had yielded an RNA enzyme called R18 that could stick 14 nucleotides – the building blocks of RNA and DNA – onto an existing RNA, using another RNA as a template (Science, vol 292, p 1319). Any self-replicating RNA, however, needs to build RNAs that are at least as long as itself – and R18 doesn’t come close. It is 189 nucleotides long, but the longest RNA it can make contains just 20.
A big advance came earlier this year, when Philipp Holliger of the MRC Laboratory of Molecular Biology in Cambridge, UK, and colleagues unveiled an RNA enzyme called tC19Z. It reliably copies RNA sequences up to 95 letters long, almost half as long as itself (Science, vol 332, p 209). To do this, tC19Z clamps onto the end of an RNA, attaches the correct nucleotide, then moves forward a step and adds another. “It still blows my mind that you can do something so complex with such a simple molecule,” Holliger says.
“It stills blows my mind that you can do something so complex with such a simple molecule”
So biologists are getting tantalisingly close to creating an RNA molecule, or perhaps a set of molecules, capable of replicating itself. That leaves another sticking point: where did the energy to drive this activity come from? There must have been some kind of metabolic process going on – but RNA does not look up to the job of running a full-blown metabolism.
“There’s been a nagging issue of whether RNA can do all the chemistry,” says Adrian Ferré-D’Amaré of the National Heart, Lung and Blood Institute in Bethesda, Maryland. RNA has only a few chemically active “functional groups”, which limit it to catalysing just a few types of chemical reaction.
Functional groups are like tools – the more kinds you have, the more things you can do. Proteins have many more functional groups than RNAs. However, there is a way to make a single tool much more versatile: attach different bits to it, like those screwdrivers that come with interchangeable heads. The chemical equivalents are small helper molecules known as cofactors.
Proteins use cofactors to extend even further the range of reactions they can control. Without cofactors, life as we know it couldn’t exist, Ferré-D’Amaré says. And it turns out that RNA enzymes can use cofactors too.
In 2003, Hiroaki Suga, now at the University of Tokyo, Japan, created an RNA enzyme that could oxidise alcohol, with help from a cofactor called NAD+ which is used by many protein enzymes (Nature Structural Biology, vol 10, p 713). Months later, Ronald Breaker of Yale University found that a natural RNA enzyme, called glmS, also uses a cofactor.
Many bacteria use glmS, says Ferré-D’Amaré, so either it is ancient or RNA enzymes that use cofactors evolve easily. Either way, it looks as if RNA molecules would have been capable of carrying out the range of the reactions needed to produce energy.
So the evidence that there was once an RNA world is growing ever more convincing. Only a few dissenters remain. “The naysayers about the RNA world have lost a lot of ground,” says Donna Blackmond of the Scripps Research Institute in La Jolla, California. But there is still one huge and obvious problem: where did the RNA come from in the first place?
RNA molecules are strings of nucleotides, which in turn are made of a sugar with a base and a phosphate attached. In living cells, numerous enzymes are involved in producing nucleotides and joining them together, but of course the primordial planet had no such enzymes. There was clay, though. In 1996, biochemist Leslie Orgel showed that when “activated” nucleotides – those with an extra bit tacked on to the phosphate – were added to a kind of volcanic clay, RNA molecules up to 55 nucleotides long formed (Nature, vol 381, p 59). With ordinary nucleotides the formation of large RNA molecules would be energetically unfavourable, but the activated ones provide the energy needed to drive the reaction.
This suggests that if there were plenty of activated nucleotides on the early Earth, large RNA molecules would form spontaneously. What’s more, experiments simulating conditions on the early Earth and on asteroids show that sugars, bases and phosphates would arise naturally too. It’s putting the nucleotides together that is the hard bit there does not seem to be any way to join the components without specialised enzymes. Because of the shapes of the molecules, it is almost impossible for the sugar to join to a base, and even when it does happen, the combined molecule quickly breaks apart.
This apparently insurmountable difficulty led many biologists to suspect to RNA was not the first replicator after all. Many began exploring the possibility that the RNA world was preceded by a TNA world, or a PNA world, or perhaps an ANA world. These are all molecules similar to RNA but whose basic units are thought to have been much more likely to form spontaneously. The big problem with this idea is that if life did begin this way, no evidence of it remains. “You don’t see a smoking gun,” says Gerald Joyce, also of the Scripps Research Institute.
In the meantime John Sutherland, at the MRC Laboratory of Molecular Biology, has been doggedly trying to solve the nucleotide problem. He realised that researchers might have been going about it the wrong way. “In each nucleotide, you see a sugar, a base and a phosphate group,” he says. “So you assume you need to make those building blocks first and then stick them together… and it doesn’t work.”
Instead he wondered whether simpler molecules might assemble into a nucleotide without ever becoming sugars or bases. In 2009, he proved it was possible. He took half a sugar and half a base, and stuck them together – forming the crucial sugar-base link that everyone had struggled with. Then he bolted on the rest of the sugar and base. Sutherland stuck on the phosphate last, though he found that it needed to be present in the mixture for the earlier reactions to work (Nature, vol 459, p 239).
The prion: the infectious agent
Some prion disease appear to be infectious. That is, one can isolate something from an infected individual, give it to another individual and that individual will get the disease and make more of the infectious material. This is the behavior one expects for an infectious agent, such as a virus or bacterium. (Microbiologists would say that the prion infectious agent satisfies Koch's postulates, a set of groundrules used to show that one has an infectious agent.)
So what kind of an infectious agent is it? This is the step at which the biologists get very fascinated with the prion. The properties of the infectious agent do not correspond to those of any known agent. In particular.
* The prion agent is not inactivated by a wide range of treatments that should inactivate viruses or bacteria.
* The prion agent, so far as we can tell, contains no nucleic acid -- no genome.
Now, prion infectious material is not easy to handle, and early experiments showing these properties were subject to challenge. However, further work continued to support these properties. Thus it seemed that the prion agent was not an ordinary agent. In fact, it almost seemed that the prion agent was a self-replicating protein. The problem is that "a self-replicating protein" does not fit with our modern understanding of proteins. "A self-replicating protein" would be a major violation of the "Central Dogma", which says that only nucleic acids can "self-replicate". This is why biologists have been fascinated by the prion agent. If it really did what it seemed to do, it would reveal a major weakness in our understanding of genes and proteins.
Two major developments have served to bring some clarity to the nature of the prion agent. We discuss these in the following two sections. At that point, we will present the current working model for the nature of the prion agent -- a model which is now widely accepted, yet has still not been clearly shown to be correct.
A plasmid is an independent, circular, self-replicating DNA molecule that carries only a few genes. The number of plasmids in a cell generally remains constant from generation to generation. Plasmids are autonomous molecules and exist in cells as extrachromosomal genomes, although some plasmids can be inserted into a bacterial chromosome, where they become a permanent part of the bacterial genome. It is here that they provide great functionality in molecular science.
Plasmids are easy to manipulate and isolate using bacteria (see also alkaline lysis) They can be integrated into mammalian genomes, thereby conferring to mammalian cells whatever genetic functionality they carry. Thus, this gives you the ability to introduce genes into a given organism by using bacteria to amplify the hybrid genes that are created in vitro. This tiny but mighty plasmid molecule is the basis of recombinant DNA technology.
There are two categories of plasmids. Stringent plasmids replicate only when the chromosome replicates. This is good if you are working with a protein that is lethal to the cell. Relaxed plasmids replicate on their own. This gives you a higher ratio of plasmids to chromosome.
So how do we manipulate these plasmids?
1. Mutate them using restriction enzymes, ligation enzymes, and PCR. Mutagenesis is easily accomplished by using restriction enzymes to cut out portions of one genome and insert them into a plasmid. PCR can also be used to facilitate mutagenesis. Plasmids are mapped out indicating the locations of their origins of replication and restriction enzyme sites.
2. Select them using genetic markers. Some bacteria are antibiotic resistant. While this is a serious health problem, it is a godsend to molecular scientists. The gene that confers antibiotic resistance can be added (ligated) to the gene you are inserting into the plasmid. So every plasmid that contains your target gene will not be killed by antibiotics. After you transfect your bacterial cells with your engineered plasmid (the one with the target gene and the antibiotic resistant marker), you incubate them in a nutrient broth that also contains antibiotic (usually ampicillin). Any cells that were not transfected (this means they do not have your target gene in them) are killed by the antibiotic. The ones that do have the gene also have the antibiotic resistant gene, and therefore survive the selection process.
3. Isolate them (such as with alkaline lysis).
4. Transform them into cells where they become vectors to transport foreign genes into a recipient organism.
There are some minimum requirements for plasmids that are useful for recombination techniques:
1. Origin of replication (ORI). They must be able to replicate themselves or they are of no practical use as a vector.
2. Selectable marker. They must have a marker so you can select for cells that have your plasmids.
3. Restriction enzyme sites in non-essential regions. You don't want to be cutting your plasmid in necessary regions such as the ORI.
In addition to these necessary requirements, there are some factors that make plasmids either more useful or easier to work with.
1. Small. If they are small, they are easier to isolate (you get more), handle (less shearing), and transform.
2. Multiple restriction enzyme sites. More sites give you greater flexibility in cloning, perhaps even allowing for directional cloning.
3. Multiple ORIs. It is important to note that two genes must have different ORIs if they are going to be inserted in the same plasmid.
It is hard to come up with a scenario where ETs would be dangerous to us
I thought I'd just talk about this briefly as Stephen Hawking has famously said we should be careful about contacting ETs in case they are dangerous.
If Earth and our solar system was attractive for them as a place to live, then the solar system would already be filled with their habitats. We wouldn't have had a chance to evolve because they would have colonized the Earth long ago, billions of years ago most likely.
As for taking our wealth, our animals or plants or ourselves - well any ET with billions of years old technology, would surely have no problem replicating anything they find of interest, any biology etc. They could create a new Stanford torus type habitat as easily as we can build a house, one especially designed just for housing their finds.
With space habs so easy to make, for them (surely), they wouldn't need the surface of the Earth at all. With their 3D printers, able to print at sub nano-scale they would also probably have universal replicators just as in Star Trek. This is not so far into our future, even 3D printers able to print food may not be far away (has already been done in the lab) - though other parts of Star Trek such as transporters, and force fields remain fantasy.
I enjoy stories about alien invasions of the Earth, am able to read them just as I read stories about magic and dragons etc, using suspension of disbelief. But once you start to think of them as creatures who are at home in space and find everything they need in comets and asteroids, there is only one story I can remember now that seems plausible, where the Aliens found things on Earth that they wanted to take away from them.
In this story, they are collectors who want to collect our art treasures, and just as collectors on Earth want to have an original Van Gogh rather than a replica you could imagine a really keen art collector who wants to have originals of our artifacts (whatever seems interesting to them). But their ideas of what is precious and collectible on Earth might not align with our ideas on these matters. Sorry I can't remember the name of the story or the author right now, maybe someone reading this knows?
You could count ET as well, taking away a few plants as collectors, is plausible, similarly, like the collectors of art, that they want to propagate the plants from originals.
It seems if they exist, at least they have strong laws or morals or directives governing their behaviour or again they would be here already as colonists.
They might turn our ideas on their heads, some of them, and so indirectly be disruptive in that way, but I doubt myself that they would be dangerous to us directly. In any case we have already announced our presence via radar and TV there is nothing we can do about that, and listening out to find out more about them seems wise whatever they are like.
How Many Cells Are in the Human Body?
All living beings are made up of cells. Some of them are made up of only one cell and others have many cells. The average adult human body has around 37.2 trillion cells. WOW, that's a lot of cells. So many, in fact, that it's hard to picture. But let's try to imagine it: If we lined up all the cells in a human body end to end, could the line reach around the Earth? If so, how many times?
An adult human body is made up of about 37.2 trillion cells. If we were able to put all of these cells end to end, how many times do you think they would circle the Earth? Click to find the answer.
Cells got their name from an Englishman named Robert Hooke in the year 1665. He first saw and named "cells" while he was experimenting with a new instrument we now call a "microscope."
A drawing of cork seen through the microscope by Robert Hooke.
For his experiment, he cut very thin slices from cork. He looked at these slices under a microscope. He saw tiny box-like shapes. These tiny boxes reminded him of the plain small rooms that monks lived in called "cells".
Vectors Used in Genetic Engineering | Biotechnology
In this article we will discuss about vectors used in genetic engineering.
By cloning, one can produce unlimited amounts of any particular fragment of DNA. In principle, the DNA isolated and cut pieces are introduced into a sui­table host cell, usually a bacterium such as Escherichia coli, where it is replicated, as the cell grows and divides.
However, replication will only occur if the DNA contains a sequence which is recognized by the cell as an origin of replication. Since such sequences are infrequent, this will rarely be so, and therefore, the DNA to be cloned, has to be attached to a carrier, or vector DNA which does contain an origin of replication.
Criteria of an Ideal Vector:
Vectors are those DNA molecules that can carry a foreign DNA fragment when inserted into it. A vector must possess certain minimum qualifications to be an efficient agent for the transfer, maintenance and amplification of the passenger DNA.
1. The vector should be small and easy to isolate.
2. They must have one or more origins of replication so that they will stably main­tain themselves within host cell.
3. Vector should have one or more unique restriction sites into which the recombi­nant DNA can be inserted.
4. They should have a selectable marker (antibiotic resistance gene) which allows recognition of transformants.
5. Vector DNA can be introduced into a cell.
6. The vector should not be toxic to host cell.
Types of Vector:
Based on the nature and sources, the vectors are grouped into bacterial plasmids, bacteriophages, cosmids and phagemids (Fig. 22.3).
Plasmids are the extra-chromosomal, self-replicating, and double stranded closed and circular DNA molecules present in the bacterial cell. A number of properties are specified by plasmids such as antibiotic and heavy metal resistance, nitro­gen fixation, pollutant degradation, bacteriocin and toxin production, colicin factors, etc.
Plasmids have following advantages as cloning vehicle (Cohen et a. 1973):
1. It can be readily isolated from the cells.
2. It possesses a single restriction site for one or more restriction enzymes.
3. Insertion of foreign DNA does not alter the replication properties.
4. It can be reintroduced into cell.
5. Selective marker is present.
6. Transformants can be selected easily by using selective medium.
7. Multiple copy numbers are present in a cell.
Some plasmid vectors are pBR 322, pBR 327, pUC vectors, yeast plasmid vector and Ti, Ri plasmids. Ti and Ri Plasmids are widely used in plant system for genetic transfor­mation.
Among higher plants, Ti plasmid of Agrobacterium tumefaciens or Ri plasmid of A. rhizogenes are the best known vectors. T-DNA, from Ti or Ri plasmid of Agrobacte­rium, is considered to be very potential for foreign gene transfer in cloning experiments with higher plants.
pBR 322 and pUC Vectors:
pBR322 is a derived plasmid from a naturally occurring plasmid col El, composed of 4362 bp DNA and its replication may be more faster. The plasmid has a point of origin of replication (ori), two selectable marker genes conferring resistance to antibiotics, e.g., ampicillin (amp r ), tetracycline (tet r ) and unique recognition sites for 20 restriction endonucleases.
Tetracycline resistance gene has a cloning site and insertion of foreign segment of DNA will inactivate the tet r gene. The recombinant plas­mid will allow the cells to grow only in presence of ampicillin but will not protect them against tetracycline .
Another plasmid used in gene cloning is pUC vector available in pairs with reverse orders of restriction sites relative to lac z promoter. This is a synthesized plasmid possess­ing ampicillin resistance gene (amp r ), origin of replication from pBR322(on) and lac z J gene from E. coli. pUC 8 and pUC 9 make one such pair.
The bacteriophage has linear DNA molecule, a single break will generate two fragments, foreign DNA can be inserted to generate chimeric phage parti­cle. But as the capacity of phage head is limited, some segments of phage DNA, not having essential genes, may be removed. This technique has been followed in λ (Lambda) phage vectors to clone large foreign particle.
Plasmid can clone up to 20 to 25 kb long fragments of eukaryotic genome. The examples of different Lambda phage vectors are λ gt 10, λ gt 11, EMBL 3, etc. M-13 is a filamentous bacteriophage of E. coli whose single stranded circular DNA has been modified variously to give rise M-13 series of cloning vectors.
Cosmids are plasmid particles, into which certain specific DNA sequences, namely those for cos sites are inserted which enable the DNA to get packed in X particle. Like plasmids, the cosmids perpetuate in bacteria without lytic develop­ment. The cosmids have high efficiency to produce a complete genomic library
These are prepared artificially by combining features of phages with plasmids. One commonly used phagemid is pBluescript IIKs derived from pUC-19.
(e) Plant and Animal Viruses:
A number of plant and animal viruses have also been used as vectors both for introducing foreign genes into cells and for gene amplification. Cauliflower Mosaic Viruses (CaMV), Tobacco Mosaic Viruses (TMV) and Gemini Virus are three groups of viruses that have been used as vectors for cloning of DNA segments in plant system. SV 40 (Simian Virus 40), human adenoviruses and retroviruses are poten­tial as vectors for gene transfer into animal cells.
(f) Artificial Chromosomes:
Yeast Artificial Chromosome (YAC) or Bacterial Artificial Chromosome (BAC) vectors allow cloning of several hundred kb pairs which may represent the whole chromosome. It can be cloned in yeast or bacteria by ligating them to vector sequences that allow their propagation as linear artificial chromosome.
Transposable elements like Ac-Ds or Mu-1 of Maize, P-element of Drosophila may also be used for cloning vector and transfer of gene among eukaryotes.
A vector that has been constructed in such a way that inserted DNA molecule is put under appropriate promoter and terminator sequences for high level expression through efficient transcription and trans­lation. Example: Use of promoters (‘nos’ from T-DNA) or expression cas­settes (pRT plasmids) (Fig. 22.3d).
The Mystery of the Minimal Cell, Craig Venter's New Synthetic Life Form
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Tom Deerinck and Mark Ellisman
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Peel away the layers of a house—the plastered walls, the slate roof, the hardwood floors—and you’re left with a frame, the skeletal form that makes up the core of any structure. Can we do the same with life? Can scientists pare down the layers of complexity to reveal the essence of life, the foundation on which biology is built?
That’s what Craig Venter and his collaborators have attempted to do in a new study published this week in the journal Science. Venter’s team painstakingly whittled down the genome of Mycoplasma mycoides, a bacterium that lives in cattle, to reveal a bare-bones set of genetic instructions capable of making life. The result is a tiny organism named syn3.0 that contains just 473 genes. (By comparison, E. coli has about 4,000 to 5,000 genes, and humans have roughly 20,000.)
Yet within those 473 genes lies a gaping hole. Scientists have little idea what roughly a third of them do. Rather than illuminating the essential components of life, syn3.0 has revealed how much we have left to learn about the very basics of biology.
“To me, the most interesting thing is what it tells us about what we don’t know,” said Jack Szostak, a biochemist at Harvard University who was not involved in the study. “So many genes of unknown function seem to be essential.”
“We were totally surprised and shocked,” said Venter, a biologist who heads the J. Craig Venter Institute in La Jolla, Calif., and Rockville, Md., and is most famous for his role in mapping the human genome. The researchers had expected some number of unknown genes in the mix, perhaps totaling five to 10 percent of the genome. “But this is truly a stunning number,” he said.
The seed for Venter’s quest was planted in 1995, when his team deciphered the genome of Mycoplasma genitalium, a microbe that lives in the human urinary tract. When Venter’s researchers started work on this new project, they chose M. genitalium—the second complete bacterial genome to be sequenced—expressly for its diminutive genome size. With 517 genes and 580,000 DNA letters, it has one of the smallest known genomes in a self-replicating organism. (Some symbiotic microbes can survive with just 100-odd genes, but they rely on resources from their host to survive.)
M. genitalium’s trim package of DNA raised the question: What is the smallest number of genes a cell could possess? “We wanted to know the basic gene components of life,” Venter said. “It seemed like a great idea 20 years ago—we had no idea it would be a 20-year process to get here.”
Venter and his collaborators originally set out to design a stripped-down genome based on what scientists knew about biology. They would start with genes involved in the most critical processes of the cell, such as copying and translating DNA, and build from there.
But before they could create this streamlined version of life, the researchers had to figure out how to design and build genomes from scratch. Rather than editing DNA in a living organism, as most researchers did, they wanted to exert greater control—to plan their genome on a computer and then synthesize the DNA in test tubes.
In 2008, Venter and his collaborator Hamilton Smith created the first synthetic bacterial genome by building a modified version of M. genitalium’s DNA. Then in 2010 they made the first self-replicating synthetic organism, manufacturing a version of M. mycoides’ genome and then transplanting it into a different Mycoplasma species. The synthetic genome took over the cell, replacing the native operating system with a human-made version. The synthetic M. mycoides genome was mostly identical to the natural version, save for a few genetic watermarks—researchers added their names and a few famous quotes, including a slightly garbled version of Richard Feynman’s assertion, “What I cannot create, I do not understand.”
With the right tools finally in hand, the researchers designed a set of genetic blueprints for their minimal cell and then tried to build them. Yet “not one design worked,” Venter said. He saw their repeated failures as a rebuke for their hubris. Does modern science have sufficient knowledge of basic biological principles to build a cell? “The answer was a resounding no,” he said.
So the team took a different and more labor-intensive tack, replacing the design approach with trial and error. They disrupted M. mycoides’ genes, determining which were essential for the bacteria to survive. They erased the extraneous genes to create syn3.0, which has a smaller genome than any independently replicating organism discovered on Earth to date.
What’s left after trimming the genetic fat? The majority of the remaining genes are involved in one of three functions: producing RNA and proteins, preserving the fidelity of genetic information, or creating the cell membrane. Genes for editing DNA were largely expendable.
But it is unclear what the remaining 149 genes do. Scientists can broadly classify 70 of them based on the genes’ structure, but the researchers have little idea of what precise role the genes play in the cell. The function of 79 genes is a complete mystery. “We don’t know what they provide or why they are essential for life—maybe they are doing something more subtle, something obviously not appreciated yet in biology,” Venter said. “It’s a very humbling set of experiments.”
Venter’s team is eager to figure out what the mystery genes do, but the challenge is multiplied by the fact that these genes don’t resemble any other known genes. One way to investigate their function is to engineer versions of the cell in which each of these genes can be turned on and off. When they’re off, “what’s the first thing to get messed up?” Szostak said. “You can try to pin it to general class, like metabolism or DNA replication.”
Venter is careful to avoid calling syn3.0 a universal minimal cell. If he had done the same set of experiments with a different microbe, he points out, he would have ended up with a different set of genes.
In fact, there’s no single set of genes that all living things need in order to exist. When scientists first began searching for such a thing 20 years ago, they hoped that simply comparing the genome sequences from a bunch of different species would reveal an essential core shared by all species. But as the number of genome sequences blossomed, that essential core disappeared. In 2010, David Ussery, a biologist at Oak Ridge National Laboratory in Tennessee, and his collaborators compared 1,000 genomes. They found that not a single gene is shared across all of life. “There are different ways to have a core set of instructions,” Szostak said.
Moreover, what’s essential in biology depends largely on an organism’s environment. For example, imagine a microbe that lives in the presence of a toxin, such as an antibiotic. A gene that can break down the toxin would be essential for a microbe in that environment. But remove the toxin, and that gene is no longer essential.
Venter’s minimal cell is a product not just of its environment, but of the entirety of the history of life on Earth. Sometime in biology’s 4-billion-year record, cells much simpler than this one must have existed. “We didn’t go from nothing to a cell with 400 genes,” Szostak said. He and others are trying to make more basic life-forms that are representative of these earlier stages of evolution.
Some scientists say that this type of bottom-up approach is necessary in order to truly understand life’s essence. “If we are ever to understand even the simplest living organism, we have to be able to design and synthesize one from scratch,” said Anthony Forster, a biologist at Uppsala University in Sweden. “We are still far from this goal.”
Original story reprinted with permission from Quanta Magazine, an editorially independent publication of the Simons Foundation whose mission is to enhance public understanding of science by covering research developments and trends in mathematics and the physical and life sciences.