feasibility. One of the containers in the experiment holds a liquid that mimics the oceans of the early Earth. The water is rich in carbon dioxide and iron, has a pH of 5.5 and is held at room temperature. The other container is heated to 130oC, and its water is laden with hydrogen and sulphide with a Ph of 11. This second fluid is meant to stand in for the hot waters that spewed out of ocean-bottom springs early in the Earth's history. The liquids mix in a chrome steel pressure barrel containing a catalyst of iron and nickel sulphide. It is supposed to be the pores within the "chimneys" near the ocean hot springs. The gels in the chimneys act as membranes allowing small molecules such as nutrients and wastes to pass through but keeping the macro-molecules such as proteins inside. His goal is to demonstrate that the

Figure 11-04g2 Origin of Life Experiment

experiment can produce amino acids and peptides. He has yet to reproduce life's first steps of making simple organic molecules like methane and acetate.

(b) RNA World -

Starting in 1991, biochemist Jack Szostak (and his graduate students) has embarked on a project to recreate life in the laboratory from where Stanley Miller left off. Followings is a summary of their accomplishments up till 2004:


1. By a process of artificial selection, a ribozyme (an enzyme made from RNA instead of protein) was created to copy up to 14 nucleotides from an RNA with an accuracy of roughly 97%. (By 2013 it is reported that the 202 nucleotides long RNA enzyme can produce 206 nucleotides long RNA at -17^oC)
2. Fatty acids were used to form bubbles (for cell membranes) known as vesicles. These vesicles could grow and divide as they were forced through 100-nanometer-wide pores - an "early earth" simulation of the pores in rocks around hydrothermal vents. It is also found that by adding a kind of clay known as montmorillonite to the solution of fatty acids, it sped up the rate of vesicle formation 100-fold.
3. It was discovered that in a mixture of RNA, fatty acids and clay, the clay can cause nucleotides to spontaneously assemble

themselves into RNA, which is then automatically trapped inside the fatty acid bubbles. The result is something that resembles a cell: It has genetic material and water contained within a waterproof fatty-acid pouch. Figure 11-04h is an image of these makeshift cells taken with an optical microscope and enhanced using fluorescent dye, it reveals yellow bits of RNA inside spherical green vesicles. They have not created life yet. It will be their next step to assemble a system of ribozyme, RNA and vesicle. This system has to grow, divide, and evolve in order to make the transition to life. Once there's one example of a lab system that's evolving by itself, then the challenge is to build systems that can evolve under different conditions such as the high-pressure liquid hydrogen in Jupiter and Saturn. By the summer of 2007, Jack Szostak predicts that within the next six months, scientists will be able to create a cell membrane using fatty acids.

Figure 11-04h Proto-cell [view large image]

He is also optimistic about getting nucleotides to form a working genetic system. The idea is that once the container is made, if scientists add nucleotides in the right proportions, then Darwinian evolution could simply take over. A June 2008 publication by him indicates that early proto-cells

with fatty-acid-based membrances could not have been autotrophs (with nourishment generated within). Such early cells were more like the heterotrophic model, which emerged from very simple cellular structures within a complex environment that provides external sources of nutrients and energy.

The experiment of making organic molecules out of inorganic substances by Stanley Miller in 1953 is not his only origin of life investigation (Figure 11-04j). He had also filled a batch of vials in 1972 with a mixture of water (H2O), ammonia (NH3) and cyanide (any chemical compound containing the cyano group - CN), chemicals that scientists believe existed on early Earth and may have contributed to the rise of life. He had then cooled the mixture to the temperature of Jupiter's icy moon Europa (~ -170oC). Most scientists believe that it is too cold for much of anything to happen. Examination of the vials in his laboratory at the University of California at San Diego 25 years later in 1997 reveals that the normally colorless mixture had deepened to amber (Figure 11-04i) indicating the presence of complex polymers made up of organic molecules. Tests later confirmed that the mixture had coalesced into the molecules of life: nucleotides, the building blocks of RNA and DNA, and amino acids, the building blocks of proteins. It turns out that the microscopic pockets of liquid within the ice increase

Figure 11-04i Life from Ice

the concentration of the primordial soup causing the reaction rate to go up. But it was suspected that the "growth" is the result of contamination. Since then other experiments on formation of new strand of RNA, and creating RNA out of individual nucleotides had been successful under such cold environment.

Thus, the idea is influencing not just theories about life's origin on Earth but the possibility of life on Europa, Enceladus, and Titan is now more relevant than ever. Shortly after Miller finished his 25-year experiment, he suffered a stroke that ended his career. His laboratory, with 40 years of samples, was emptied in 2002 to make way for a building renovation. Experiments that had run for years or decades were discarded without ever being analyzed. Only a few items from the freezer had been rescued, the rest were incinerated for fear of cyanide poisoning. Miller was present for a few hours of this ordeal, struggling to find words to identify the vials that he had known so well. He died on May 20, 2007.

Figure 11-04j Stanley Miller [view large image]

Since then the idea of life starting in ice has inspired further investigations both out in the field north of the Arctic Circle and in the laboratories. These works provide better understandings about chemical reactions within the tiny pockets of water insider the ice (Figure 11-04k). However, this theory should not be considered as the only one in exclusion of the others. Glaciers on early Earth could have scooped up mineral dust; volcanoes could have rained ash onto nearby sea ice. Primordial ice must have been full of impurities with some organic compounds already.

Figure 11-04k Ice Catalyst

Followings are some novel properties about the icy environment as published in a special issue (in Summer 2011) on "Evolution" by the Discover Magazine.

1. Eutectic Freezing - As an ice crystal forms, only molecules of water join the growing crystal, while impurities like salt or cyanide are excluded. These impurities become crowded in the microscopic pockets of liquid within the ice and thus there are more chance for the formation of a variety of macro-molecules such as the nucleotides. The cold environment also stabilize the products by slowing down evaporation.
2. Frozen Cyanide - It is found that frozen cyanide, in the presence of ammmonia, can form a nucleotide called adenine. This is a more specific macro-molecules related to the formation of life. It is suspected that cyanide was abundant on early Earth, deposited here by comets or created in the atmosphere by ultraviolet light or lightning.
3. Ice-Water Interface - Studying of ice inside the Arctic Circle reveals that there is strong bonding between the surface of ice and water. This bonding is important for producing long organic chains like RNA. Further study in the laboratory showed that the icy environment can assemble individual nucleotides into long chain of up to 700 bases (with an RNA template). The chain would be about 30 bases long without an RNA template.
4. RNA Enzymes - At room temperature the RNA enzyme acts like scissors, snipping other RNA molecules into pieces. The process runs in reverse in the icy environment. It is found that short segments of RNA enzyme tend to join just about any kind of molecules producing chains in random sequence (not just the RNA type). The billions and billions of different arrangements eventually generated something meaningful and can replicate itself - the beginning of life.

		in kick-start key chemical reactions at the very beginning. As illustrated in Figure 11-04m, synthesis of nucleotide into chain can be destroyed by the reverse reaction of hydrolysis unless the water molecules are removed from the environment. Such environment could be realized exactly with the drying of the clay, while more nucleotides would be added in the wetting cycle. Result on one field trip reveals that chains of RNA may have grown
Figure 11-04l Life in Mud Pool	Figure 11-04m Formation of Polymer [view large image]	wrapped in blankets on concentrated sulphuric acid (in the volcanic mud), which helps to suck water molecules out of the primordial soup.

Meanwhile on Earth, there are bacteria which can survive very harsh condition on another planet. In an Earth lab, Deinococcus radiodurans (D. rad) can withstand extreme levels of radiation, extreme temperatures, dehydration, and exposure to genotoxic chemicals. They even have the ability to repair their own DNA, usually within 48 hours. Known as an extremophile, bacteria such as D. rad are of interest to NASA partly because they might be adaptable to help human astronauts survive on other worlds. A recent map of D. rad's DNA might allow biologists to augment their survival skills with the ability to produce medicine, clean water, and oxygen. Already they have been genetically engineered to help clean up spills of toxic mercury. Likely one of the oldest surviving life forms, D. rad was discovered by accident in the 1950s when scientists investigating food preservation techniques could not easily kill it. In Figure 11-04n Deinococcus radiodurans grow quietly in a petri dish.

Figure 11-04n Bacteria, D. rad [view large image]

According to Arch Mission, there would be no chance for the tardigrades to contaminate the Moon since it has no water to revive the little bugs even if they manage to get out of the enclosure somehow. That could be exactly the situation with the Beresheet crash-landing although they maintain that the library should be intact. An interesting case arises if in the next million or billion years, an ice-bearing comet hit the Moon near the crash site. The impact will melt the epoxy resins releasing the plucky tardigrades which will then flourish and turns into some kind of indestructible monster ...

Figure 11-04o Tardigrades on Moon [view large image]

It is a nice plot for science fiction or is it how multicelluar life began on Earth? i.e., via delivery from another world triggering the Cambrian Explosion.

Analysis of tardigrade's genome in 2016 reveal an unique sequence expressing the Damage suppressor protein (Dsup), which is present only in tardigrades. Further research indicates that Dsup binds to nucleosomes and protects DNA from hydroxyl radicals as well as high energy radiation. Genetically modified human cells to express Dsup also safeguards against damage from X-rays (Figure 11-04oa). It turns out that the Dsup forms a cloud-like structure; the cloud surrounds the DNA's chromatin envelope, blocking hydroxyl radicals and preventing them from disrupting cellular DNA (see insert), it works on free DNA as well. Computer simulation of Dsup in complex with DNA shows that it is an intrinsically disordered protein. Its flexibility and electrostatic charge helps it bind to DNA and from aggregates.

Figure 11-04oa Tardigrade, Protection by Dsup [view large image]

Right now, the main applications are in pharmaceuticals and crop plants. Other possibilities include cancer treatment, and space traveling.

several essential roles in cells: it maintains the structure of DNA and RNA, combines with lipids to make cell membranes and transports energy within the cell through the macro-molecule ATP. Many science-fictions portray alternate life-forms using silicon instead of carbon, but this discovery marks the first known case in a real organism. However, rigour scientific method demands that the researchers should demonstrate the presence of arsenic not just in the microbial cells, but in specific biomolecules within them. Such requirement would take much more work to satisfy. It is also known that arsenate forms

Figure 11-04p Arsenic Bacteria

much weaker bonds in water than phosphate. It breaks apart on the order of minutes. Thus they should also explain how to stabilize the AsO43- structure in such circumstance.

Further scrutiny of the arsenic data indicates that the microbe is not using the arsenic, but instead is scavenging every possible phosphate molecule while fighting off arsenic toxicity. As shown in Figure 11-04q, the cell's large vacuoles may be their way of sequestering the arsenic. It is also noted that the cells grown in high concentrations of arsenate did not seem to contain any RNA - possibly because RNA production had shut down to conserve phosphate.

Figure 11-04q Arsenic Vacuoles

As Christmas 2010 approaching, the authors of the original paper finally response to criticism by posting their supporting evidences online. One of the authors also appears in an interview to express her personal feeling about the pressure created by all the furies.

			Such field is thriving with more funding from various agents. The Synthetic Biology Journal started publication in 2016, and researchers from 17 laboratories in the Netherlands formed the group Building a Synthetic Cell (BaSyC), which aims to construct a “cell-like, growing and dividing system” within ten years.
Figure 11-04r Synthetic Life [view large image]	Figure 11-04s Synthetic Membrance [view large image]	Figure 11-04t Incorporation of Proteins [view large image]

The followings summarizes some of the recent progress in this field as reported by the afore-mentioned paper.



Cell Membrance - It is well known that macromolecules such as phospholipid can naturally form a 3-D sphere bound by a membrane in water (the droplet is called liposome, Figure 11-04u). In anticipation of making the cell to divide, the surface or its interior was laced with Min proteins which generate some sort of oscillation to specify the mid-zone of the a divdiding cell. Initial problem of instability was overcome by special setup such as the one shown in Figure 11-04s. The researchers then experimented with pressurizing, deforming and reshaping the liposomes to take on non-spherical shapes that mimic cells better. They also designed a chip that could mechanically split a liposome in two by pushing it up against a sharp point. It is recognized that this is not cell division in real life.

Figure 11-04u Cell Membrance

BTW, microfluidics chip is used to study the behaviour of fluids through micro-channels.

Metabolism - There may be some chemical reactions occuring in a bag of randomly gathered macromoleucles (Figure 11-04v), but the process would be very slow and would come to a halt without suitable enzymes and energy infusion. It is also found that some components need to be kept apart, while others brought together in real life. Rudimentary synthesis of life just load proteins into the vesicles using microfluidic channel as shown in Figure 11-04t. They managed to load ATP synthase into the vesicle and try to mimic the production of ATP to supply bio-energy as in real cell. Another group is interested in pathways that allow photosynthetic microbes to pull carbon dioxide from the environment and make sugars and other cellular building blocks. They found 17 enzymes from 9 different organisms to perform the task getting only an inefficient and slow reaction so far.

Figure 11-04v A Bag of Chemicals

Other example involves encapsulating the motor protein kinesin and the microtubule to produce a force-generating network that can propel the movement of the vesicle.

• Genes -The next step is to make the cell to replicate. Such research has been running by JCVI since 2006. The methodology requires a lot of help from living bacteria - it is not really synthetical. See "Minimal Genome" for detail.
  
  

  Bioreactor is the fancy name for the process of fementation (Figure 11-04x). While bacteria are used to make cheese for hundreds of years, the modern version uses them to produce biochemicals for drug, sewage treatment, tissue engineering, ... A side benefit in the search for synthetic life is the discovery that by introducing genetic circuits into the liposomes, new proteins can be produced to provide novel solutions for treating cancer, tackling antibiotic resistance or cleaning up toxic sites, ...

  Figure 11-04x Bioreactor

		controls can easily be incorporated into the process or a kill switch can be installed to render the things harmless. Anyway, Figure 11-04z shows the trailer of a 1969 movie: "The Andromeda Strain" (Andromeda stands for "A" - the first case of outer space attack, the microbes are not from Andromeda) in which a satellite brought back deadly microbes from outer space ...
Figure 11-04y Crossota Millsae	Figure 11-04z Andromeda Strain [view large image]

¹The Murchison meteorite was recovered in Australia in 1969. Analysis of the meteorite found over 90 types of amino acids as well as some left-handed sugar that does not exist naturally on Earth. This rare form of substance tends to prove the extraterrestrial origin of the rest of the contents. The Murchison meteorite contains the same amino acids obtained by Stanley Miller in his laboratory, and even in the same relative proportions

²Metabolism is the sum of all chemical activities occurring inside a living cell. Metabolic cycle (pathway) begins with a particular reactant and terminate with an end product.

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