Archive for the “Biotechnology” Category

What could those three possibly have in common? Believe it or not, they all play the role of a lead action figure in kids video games. Hearing the latest edition of the german biotechnology news broadcast, I was surprised to learn that researchers at the Riedel-Kruse Lab in Stanford University have developed, what-they-call, Biotic Video Games, where the paramecium are controlled and maneuvered about via a joystick and managed to publish their findings in a research paper!

The device is basically composed of a fluid compartment, where the paramecium move around and roam about freely. I am sure you are wondering, exactly how BIG (small) IS a paramecium. Well, it is so small, making it actually difficult to observe by the naked eye. But no worries! They can be seen quite clearly on your screen, while you’re playing, thanks to the provided microscope camera, which is connected to electrodes and supplies you with a live feed, being superimposed on the flash game board onto your screen. The joystick is capable of creating a weak electric field, which influences the direction of their movement, as you wish.

Eight different games have been developed and given quite funny names, as Ciliaball, Pac-man and Pond Pong. For instance, in one game version, the player needs to move about the paramecium to score a soccer goal. To help you easier imagine this, take a look at this 3-part video.

As Riedel-Kruse put it, these games serve two ultimate goals: First, to awaken the scientific interest in those young kids and teenagers, hopefully motivating them to someday pursue a career, heading off in that direction. And after all, scientists can collect and analyze information about those tiny organisms, whilst playing with them.

So please do take a break and enjoy some time away with your paramecium ūüôā

Image Source: Stanford University Schools of Medicine and Engineering

ResearchBlogging.org Riedel-Kruse IH, Chung AM, Dura B, Hamilton AL, & Lee BC (2011). Design, engineering and utility of biotic games. Lab on a chip, 11 (1), 14-22 PMID: 21085736

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If you have ever been one of the unlucky ones waiting for a cancer diagnosis biopsy, or having a friend or a relative undergoing the process, then you must know how the wait is nerve wrecking. The standard procedure is using a biopsy needle to extract some cells of the tissue suspected for cancer, and then waiting for about a week, until the results come out. To make matters worse, results can sometimes be inconclusive or 100% correct.

Simple smartphone applications might be able to rapidly diagnose cancer in the future

Fortunately, a group of scientists at Massachusetts General Hospital (MGH) in Boston, were able to develop a new technology, that is much more rapid and almost 100% accurate in the diagnosis process. They developed a small NMR device (detects compounds by the mode of oscillation of their nuclei in a magnetic field) the size of about a coffee cup, and they were also able to synthesize magnetic Nanoparticles, which stick to certain tumor specific proteins. So now all I have to do, is head to the clinic, have the needle biopsy performed and the cells taken. Then they are mixed with the magnetic Nanoparticles and the results are taken from the small NMR device and read using a simple smartphone application.

This technique was used in the first trial on 50 patients, taking less than an hour to diagnose each. Also, as the device can detect 9 tumor associated proteins, combining the results for 4 of these gave accurate results in 48 out of the 50 patients. In another trial, the accuracy was 100% in the 20 patients tested. The conventional tests’ average accuracy is 74-84 %.

This new technology will also cut down on the cost of repeat biopsies, which can be very expensive, and scientists hope it will have many other applications as well, like patient cancer follow-up, through quantitative analysis of the tumor associated proteins. Maybe the biopsy will not be needed in the future and a simple blood test will also do….

Source: Science/AAAS

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Metagenomics is a culture independent approach that has contributed extensively to the study and understanding previously unidentified microbial communities. Seeking a further understanding of employing metagenomics in the study of the Red Sea microbial communities, we are pleased to interview Dr. Rania Siam, an Associate Professor in the Biology Department, the Director of the Biotechnology Graduate Program at the American University in Cairo (AUC) and an Investigator in the Red Sea Marine Metagenomics Project that is currently running at the AUC in collaboration with King Abdullah University of Science and Technology (KAUST), Woods Hole Oceanographic Institution and Virginia Bioinformatics Institute at Virginia Tech. Dr. Siam holds a Ph.D. in Microbiology and Immunology from McGill University. In addition, she held several post-doctoral positions at McGill Oncology Group, Royal Victoria Hospital, The Salk Institute for Biological Studies and The Scripps Research Institute. Since 2008, Dr. Hamza El Dorry (PI) and Dr. Siam (Co-PI) have been leading the Red Sea Metagenomics research team. The team is exploring novel bacterial communities in the Red Sea through actively participating in Red Sea expeditions for sampling and performing extensive molecular biology, genomics and computational analysis of the data.


1- Dr. Siam, thank you very much for accepting our request. Would you please explain for us the driving motives behind doing metagenomics research in the Red Sea?

The Red Sea is a unique environment in the region that remains to be explored. Thus, working on the Red Sea gives us the opportunity to perform essential research for the region. Furthermore, our main interest are the Red Sea brine pools that are unique environments in terms of high temperature, high salinity, high metal contents and low oxygen. Microbial communities living in these environments are known as extremophiles. The survival of extremophiles in such drastic conditions indicates their possession of genes with novel properties that underlie these unique survival characteristics. Thus, we are highly motivated to explore these novel microbial communities and their unique properties. In addition, we are interested in extracting biotechnological products from the Red Sea that can be beneficial as antimicrobials and anticancer agents.

2- Would you please outline for us the objectives of the project and the main activities inside and outside the laboratory?

In our project, one of our main aims is establishing a Red Sea marine genomic database to be accessed by scientists’ worldwide. Additionally, we are screening this database for biotechnological pharmaceutical products as enzymes and anticancer agents. There are three main activities in the project: sampling, molecular biology/genomics work and computational analysis of the data. Concerning sampling, it is a challenging process that requires rigorous planning where samples should be subjected to proper processing and storage till arrival to the labs. In labs, samples are subjected to different procedures starting from DNA extraction followed by Whole Genome Sequencing (WGS) to identify unknown genes or unique ones and help us understand novel microbial communities. This requires rigorous computational analysis to make sense of our data. Furthermore, we construct fosmid libraries for isolation and purification of genes of biotechnological interest as lipases and cellulases. In addition, we carry out 16s rRNA phylogenetic analysis on the microbial communities present in the samples.

3- Since the idea is novel, we would like to know about the nature of samples, the parameters and the challenges imposed during the process of sampling.

Basically, the sampling process requires well-equipped research vessels as the Woods Hole Oceanographic Institute ‚ÄėOceanus‚Äô and The Hellenic Center for Marine Research (HCMR) ‚ÄėAegeo‚Äô. In addition, it is essential to have a team of physical oceanographics for adequate sampling.

http://www1.aucegypt.edu/publications/auctoday/AUCTodayFall09/Cure.htm
Image Source: AUC Today

We started with two different brine pools: Atlantis II Deep and Discovery Deep. As brine pools, these two regions are characterized by the presence of extremely harsh conditions as I mentioned before. In the 2008 and the 2010 KAUST Expedition to the Red Sea, we collected two forms of samples: Large volume water and sediments. In both cases, we face challenges during sample collection. Collecting large volume water samples can take up to 4 hours. In case of bad weather, it is nearly impossible to collect samples. Regarding the sediments, the heavy weight of the sediment core is the main challenge since it may drag people to the water during the sampling process.

The water samples are collected using CTDs (an acronym for Conductivity, Temperature and Depth), it is formed of 10 liter bottles that collect samples and measure the conductivity, temperature and depth, in addition to other parameters. CTDs are capable of measuring these physical properties from each meter of water. Accordingly, they are able to retrieve 2200 readings for each parameter at 2200 m depth. This is very beneficial as it allows us to correlate the physical and chemical parameters with the nature of microbial communities obtained from each sample.

4- What are the reasons behind the choice of Atlantis II Deep and Discovery Deep brine pools for study?

These sites are unique. Atlantis II Deep is 2200 meters below the water surface. It is characterized by having high temperature (68 ¬įC), high heavy metal content, high salinity and little oxygen. Thus, these extremely drastic conditions are motivating us to explore the extremophilic microbial communities in this pool. Discovery Deep is adjacent to Atlantis II but the conditions are less harsh and varies in its heavy metal content. This encourages us to undergo comparative genomic analysis between the two regions.

5- Did the relatively new metagenomic approach of sequencing multiple genomes, combined with, the novelty of employing this approach in the study of microbial milieu in the Red Sea imply some unprecedented practical challenges in retrieving entire and authentic DNA sequences?

Yes, many challenges are present. For example, in case of sediments, many cells die. This in turn can make the process of DNA extraction more difficult. However, we managed to cope with this problem by quickly extracting the DNA from the sediments following its arrival to the labs and avoid freezing and thawing. Another problem with sediments is the presence of impurities that are co-extracted with DNA and interfere with the analysis. Concerning water samples, the main challenge is that the amount of DNA in the samples is very minute. This was dealt with by filtering large volumes of water up to 500 liters per sample.

6- Is recognizing and validating sets of data that are pointing out to specific patterns of microbial diversity or novel genes considered to be challenging?

Actually, we have retrieved enormous amount of data and a large percentage of the data has no match to sequences present in the other genomic databases. This has inspired us to think of new approaches for data analysis to identify the role of these novel sequences and seek collaborations with computational biologists.

7- Finally, do you think there are other environments in Egypt with unique properties which make it promising for employing metagenomics to discover novel genes and bacterial strains?

Yes, actually Egypt is very rich in environments with unique properties. For example, we have the deserts, Siwa‘s hot springs and the Nile. Many unique environments are yet to be explored and lots of research needs to be performed. Our natural resources are limitless.

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This¬†post on Microbe World entitled: “Software for Programming Microbes” did attract my attention, and I followed its source in the¬†MIT Technology Review:¬†An amazing article presenting a¬†fascinating¬†newly emerging technology of programming bacteria to do whatever we want them to do, produce drugs more efficiently, clean up oil spills, anything… useful! But the methodology was quite beyond my imagination.

I was extremely lucky to be able to interview Dr. Christopher Voigt, Associate Professor at the University of California, San Francisco, who is the project leader. Dr. Voigt has published over 34 articles indexed in Pubmed and you can find more about his projects on his lab website.

Now I will leave you with the interview! I thank Radwa for reviewing it.

Dr. Christopher Voigt

Dr. Christopher Voigt

1. May you please simplify the term “genetic circuit” to the micro-readers? What drove you to use software to genetically modify bacteria?
A genetic circuit functions like an electronic circuit, but uses biochemical interactions to do the computation. I am a computer programmer at heart and find living cells to be the ultimate challenge.
2. We used to hear a lot about the use of genetically modified bacteria in cleaning up toxicants or oil spills, producing drugs and biofuel. How is “programming bacteria” different from the “regular” definition of genetic engineering, which might be based on inserting a gene, a regulatory gene, or an operon that encodes for a certain needed functionality?
Genetic programming controls the timing and conditions under which those processes occur. ¬†It doesn’t refer to the pathways by which molecules are made or degraded.
3. In MIT Technology Review, you mentioned that like for a computer, programming bacteria is about writing a program to be encoded on a piece of DNA to implement a function. How can bacterial cells understand the code? How can the software make them sense the outer media?
The DNA contains codes for when molecules like proteins and mRNA should start and stop being produced and under what conditions. A protein can change its state when it senses a condition and bind to DNA to cause genes to be turned on or off.  This acts like a sensor.
4. Honestly, I can’t imagine writing a piece of code to link bacterium to one another, because node->pRight!=NULL ? I just can’t imagine it. Is there any risk of overloading natural functions by accident?
No.
5. How can programing bacteria make use of quorum sensing?
Quorum signaling enables cells to be programmed to communicate with each other.
6. How can drug discovery and production benefit from programmable bacteria in the near future?
It makes it easier to access and control those pathways.
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Forget about the old Petri dishes and culture media! A brilliant new method for the growing of microorganisms in order to study their behavior, especially in a large community, again tracking the phenomenon of quorum sensing , has been developed and put to use.

The new invention, by Connell et al., resembles a trapping sack for microorganisms, made of bovine serum albumin covalently cross-linked by laser lithography to form a three dimensional structure. These harboring chambers are very small, with a 2 to 6 picoliter capacity, and are permeable, and thus allowing an infinite influx of nutrients and other essential small molecules for the bacteria growing inside.

Scientists have already compared the growth rates of Pseudomonas cells in ‚Äúthe trap sacks‚ÄĚ to those in conventional culture media and mouse lungs and the results were promising! The new technique allows them to study patterns of antibiotic resistance, infection and biofilm formation more clearly and in earlier phases of bacterial growth‚Ķ

Source: Science magazine, Vol. 330 issue 6004.

Image source: Microbiology Bytes

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The pursuit of renewable sources of energy just hit a crucial breakthrough. Since the stores of fossil fuel are diminishing as we speak, researchers are trying to exploit the machinery of microorganisms for the production of diverse chemical compounds, which can be consumed by themselves or later channeled into some form of combustible fuel. One major group of those compounds are alkanes, those saturated organic compounds abundantly found in gasolline.

The study started off when ten out of the eleven strains of Cyanobacteria, that were¬†photoautotrophically cultured, produced forms of alkanes, mostly those with 15 & 17 carbons atoms ‚Äútermed penatdecane and heptadecane respectively‚ÄĚ. Logically, that indicates that the ‚Äėalkane-producing gene‚Äô is shared in all ten of them, yet absent in that unlucky 11th strain. So the search was launched.

Trying to pinpoint the gene responsible for the production of alkanes through using a method referred to as subtractive genome analysis, the study authors compared ten genomes of the alkane-producing strains to figure out which genes they have in common. Next, any of those shared genes was immediately eliminated if it had additionally showed up in the genome of the NON-alkane producing strain. Eventually, the researchers were left with 17 genes found in common and the function of 10 of them had already been previously assigned. Through careful scrutiny of the families to which proteins of those remaining 7 genes probably belong to, two of them particularly stood out, being likely participants in the pathway of the alkane synthesis.

And as always, there is no better way to test the hypothesis than to consult a microbiologist’s favorite lab microbe. To our pleasant surprise, extracts from the colonies of Escherichia coli engineered to express both genes did in fact contain alkanes. So, although we are still not fully aboard the track heading the way towards large scale production of alkanes using such microbes, this is definitely a gigantic leap in the right direction!

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Hansenula polymorpha, also known as Pichia angusta, with its metabolism highly dependent on methanol as a carbon source, has been excessively employed in the production of therapeutic proteins for the last two decades. The yeast was first discovered in 1950s in spoiled orange juice.

Image source:

http://www.drugdevelopment-technology.com/contractors/contract_research/artes/

The biotechnological interest in Hansenula is mainly attributed to its unique capabilities underlied by its rare characteristics. For instance, being one of the limited group of yeast that are able to assimilate methanol, gives it the advantage of being able to utilize relatively cheap substrates. In addition, Upon high temperature,  Hansenula polymorpha shifts its biochemical methanol metabolism pathway to the biosynthesis of trehalose which is a thermo-protective sugar, this fact explains its unique ability to resist temperatures up to 49 degree Celsius. further, Hansenula is able to secrete the protein products directly to the culture, a fact that renders the whole process of downstream processing easier and less costly. finally, the ability to survive in wide pH range,from 2.5 to 6.5, makes it a versatile protein factory which is exploited in the production of various types proteins, each of which requires a very different optimum pH value throughout the fermentation process.

Kg2-e.jpg

Image source:

http://en.wikipedia.org/wiki/Hansenula_polymorpha

( Budding Hansenula cells)

However, with Hansenula polymorpha post-translational modification processes are not highly regulated, this makes Hansenula¬†useful for the production of relatively small to¬†medium sized polypeptides as Parathyroid hormone, Staphylokinase, Elafin……etc.¬†¬†However, Regarding large polypeptides, mammalian cells with tightly regulated post-translational modification processes¬†represent a better option.

On the industrial scale, different strains of Hansenula polymorpha are being exploited as expression systems. For instance, a genetically engineered strain known as super-transformed strain bearing additional two advantages than the wild type strain. Being super-transformed means that it is capable of secreting Calnexin which is a protein Chaperone that functions to ensure proper folding of the secreted protein. Additionally, Calnexin enhances the protein secretion efficiency of Hansenula . Accordingly, the super-transformed strain gains its industrial potential in terms of quality assurance of the secreted protein product as well as increasing the  cost effectiveness of the industrial process.

References: 1.‚ÄúHansenula polymorpha‚ÄĚ wikipedia, www.wikipedia.org

2. Gellissen G, ‚ÄúHansenula ploymorpha : Biology & Applications‚ÄĚ, 2002

3.Marcos A. Oliveira, Victor Genu, Anita P.T. Salmazo, Dirce M. Carraro and Gon√ßalo A.G. Pereira1 ‚ÄĚThe transcription factor Snf1p is involved in a Tup1p-independent manner in the glucose regulation of the major methanol metabolism genes of HansenulaPolymorpha‚ÄĚ, Genetics and Molecular Biology, 521-528 (2003)

4. Giuseppinia Parpinello, Enrico Berardi, and Rosanna Strabbioli,‚ÄĚ A Regulatory Mutant of Hansenula polymorpha Exhibiting Methanol Utilization Metabolism and Peroxisome Proliferation in Glucose‚ÄĚ, J Bacteriol. 1998 June; 2958‚Äď2967.
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Yediot Ahronot (Literally: Latest News)

Cure for radiation sickness found?

Published: 07.17.09

A team of scientists has succeeded in developing an anti-radiation. “The process that led up to the medical innovation dates back to 2003, when Professor Gudkov ‚Äďthe head of the team- came up with the idea of using protein produced in bacteria found in the intestine to protect cells from radiation.Mice received that purified protein survived the amount of radiation that killed the control group.

What kind of news is that?! Do that legendary bacteria and that miraculous protein actually have names?! If so, why don’t newspapers include it? Should bloggers do everything?!

Professor Andrei Gudkov ‚Äď Chief Scientific Officer at Cleveland BioLabs, is interested in protecting cells against apoptosis induced by cancer therapy as well as radiotherapy. He worked on p53 ‚Äďthe famous tumor suppressor- and found that p53 has a role in inducing apoptosis. The research group suggested that p53 inhibitors can protect normal cells against chemo- and radiotherapy, and it’s been found that it sensitizes tumor cells to the therapy (PMID: 15865929). They also showed that PFTmu (pifithrin-mu), a small isolated inhibitor of p53, protected primary mouse thymocytes from p53-induced apoptosis caused by radiation (PMID: 18403709).

The breakthrough discovery mentioned above has been published in Science – 11 April 2008. It is about the injection of “flagellin” purified from Salmonella enterica serovar Dublin into mice and monkeys. It causes suppression of apoptosis by binding to Toll-like receptor 5 (TLR5) and activation of the nuclear factor‚ÄďkappaB (NF-kappaB) pathway, the same mechanism used by tumor cells to inhibit the function of the p53 pathway (PMID: 18403709). To reduce its immunogenecity and toxicity, they engineered a polypeptide derived from flagellin with the “important” domains only, N and C termini separated by a linker. The engineered protein (named: CBLB502) was found to provide radioprotection in rhesus monkeys and mice against lethal doses of gamma-radiation and accompanied hematopoietic system and gastrointestinal tract acute radiation syndrome, with no alteration of the efficacy of the radiotherapy.

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When you hear/ read the term “Phage Therapy“, you’ll be automatically directed to the concept of using bacteriophages, the virus-like particles that infect bacteria, to kill/ lyse the resistant bacterial strains, instead of the “useless” antibiotics that allowed bacteria to fool them & develop resistance against them. The initial target of phage therapy was to kill the bacteria using phages; because they act like any other virus; get in,¬†multiply and lyse the cell. But, by this way, bacteria develop resistance against phages more rapidly. So, they may become useless by time. In this paper from PNAS: “Engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy,” two bioengineers, Timothy K. Lua and James J. Collins, from Boston University successfully engineered the Enterobacteria filamentous phage M13 to weaken bacteria not to kill it. Sounds strange, right? By engineering M13, they gave us a variety of options:

1st, we may make M13 overexpress a bacterial protein named lexA3 which inhibits the ability of the bacteria to repair their damaged DNA by the action of Ofloxacin ‚Äďas pharmacophils, who had 2 consecutive chemotherapeutics courses, we may recall that quinolones’ MOA is generation of ROS. So, the repressor suppresses the bacterial SOS mechanism. Very promising results were observed; the adjuvant therapy increased the survival rate of mice infected with resistant E. coli. It was also observed that the adjuvant therapy reduced the rate of developing mutations/ resistance within the E. coli population.

Schematic of combination therapy with engineered phage and antibiotics

2nd, bacteriophage can be responsible for expression of certain proteins that can attack gene networks in bacteria which are not target for existing antibiotic classes. I will mention just one example here, expression of CsrA which is a “global regulator of glycogen synthesis and catabolism, gluconeogenesis, and glycolysis, and it also represses biofilm formation,” biofilms is thought to be related to antibiotic-resistance and OmpF porin which is used by quinolones to enter the bacterial cell, it may enhance its entrance.

Engineered phage producing both CsrA and OmpF simultaneously (csrA-ompF) enhances antibiotic penetration via OmpF and represses biofilm formation and antibiotic tolerance via CsrA to produce an improved dual-targeting adjuvant for ofloxacin

Now, thanks to the engineered phages, we can use the old beloved antibiotic classes to treat bacterial infection using the engineered phages as an adjuvent therapy to potentiate the cidal action of the antibiotic on the former-resistant strains. A precaution was made to ensure that no lysogeny would take place in the human cells is that the phages were engineered to be “nonreplicative”. But we still have two problems regarding Phage Therapy in general: identifying the strain responsible for the infection & making sure that the human immune system won’t elicit an immune response against phages, they’re “foreigners” after all!

Image credits:

1- “Schematic of combination therapy with engineered phage and antibiotics. Bactericidal antibiotics induce DNA damage via hydroxyl radicals, leading to induction of the SOS response. SOS induction results in DNA repair and can lead to survival. Engineered phage carrying the lexA3 gene (lexA3) under the control of the synthetic promoter PLtetO and an RBS acts as an antibiotic adjuvant by suppressing the SOS response and increasing cell death”: http://www.pnas.org/content/106/12/4629.figures-only

2- “CsrA suppresses the biofilm state in which bacterial cells tend to be more resistant to antibiotics. OmpF is a porin used by quinolones to enter bacterial cells. Engineered phage producing both CsrA and OmpF simultaneously (csrA-ompF) enhances antibiotic penetration via OmpF and represses biofilm formation and antibiotic tolerance via CsrA to produce an improved dual-targeting adjuvant for ofloxacin”: http://www.pnas.org/content/106/12/4629.figures-only

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Sept 16th:
Hi, Diary;
Now it’s in the press. It’s humiliating. In Scientific American, it was written under the title: Turning Bacteria into Plastic Factories. So that’s what we, E. coli, turned into: slaves for humans to do whatever they want us to do.

I remember this like it happened yesterday. It was someday in the beginning of the year 2008. That day we found ourselves in those tanks, large ones, filled with sugar & water. “This is great; we can ferment the whole amount of sugar in the tank. This is the perfect place to live in,” that is what we thought. Then we got this strange order from our genomes or plasmids, I’m not sure & found ourselves producing that enormous amount of 1, 4- Butanediol (BDO). This was so strange because BDO used to be toxic to us at low levels (I’ve heard once that any production of a non-native material inhibits our growth). Then we realized the truth; we’ve been genetically engineered to tolerate BDO, the raw material for a very large number of plastic, rubber and fiber products including solvents, fine chemicals, pharmaceuticals, automotive components, electrical and electronics components, as well as apparel fibers.

Honestly, I don’t know. Sometimes I feel that bioengineers from San Diego-based Genomatica, Inc. are right after all; they modified us, they wanted to make use of us, they wanted to save money & energy needed to produce BDO from non-renewable petrochemical feedstocks, the currently used method. Plus it causes us no harm as Bioengineer Christophe Schilling, president and co-founder of the company said: “We have engineered the organism such that it has to secrete that product in order for it to grow.”

Now I know what it feels like with my fellow bacterial species, the natural born producers. Most bacteria synthesize the organic polyesters Polyhydroxyalkanoates (PHAs) to be used as a carbon & energy storage material. Now they’re discussing the ability to use these PHAs as biodegradable plastics.

Genetically engineered E. coli may produce plastics - Scientific AmericanSource: A piece of a plastic notebook found in the Petri dish appears in the picture below, Genomatica labs, San Diego.

Image credits:
Genetically engineered E. coli may produce plastics: http://www.sciam.com/

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