A model organism – the virtual bacteria.

stanfordmycoplasmagenitaliumI was reading an article in Scientific American today that got me thinking about the complexities of biology – the article described the production of a virtual bacteria using computing to model all of the known functions of a simple single cell.  The article was a very compelling read and presented a rational argument of how this could be achieved, based on modelling of a single bacterium that would eventually divide.  The benefits of a successful approach to achieving this situation are immense for both healthcare and drug development, but the difficulties are equally immense.

In order to simplify the problem the organism chosen to be modelled is a bacterium with the smallest genome size – Mycoplasma genitalium a bacteria that has only 500+ genes all of which have been sequenced.  This bacterium is also medically important, which adds weight to the usefulness of a virtual organism.  The problem of programming the computer was divided into modules, each of which will describe a key process of the cell all of which could feedback in a way that described actual binding coefficients for protein-protein and protein-DNA interactions.

As I read the article, I began to realise that there were some simple problems associated with the description of how the computer would “manage” the cell and when the author described doubling protein concentrations prior to cell division I knew there were problems with their model – this simplistic approach is not what happens in the cell – cellular control is an important aspect of this modelling and must be correct if the cell is to be realistic.  I can illustrate what I mean with one example – plasmid replication.  A plasmid in an autonomous circle of DNA, often found in bacteria, that are easy to understand and ubiquitous in nature.

Replication of plasmid DNA:

ReplicationThe number of copies of a plasmid are tightly controlled in a bacterial cell and this control is usually governed by an upper limit to an encoded RNA or protein, when the concentration of this product drops, such as AFTER division of the cell, replication will occur and the number of plasmids will increase until the correct plasmid number is attained (indicated by the concentration of the controlling gene product).

This is a classic example of cellular control and is a lot different to a model based on doubling protein concentration in anticipation of cellular division.

Mycoplasma genetics and the complexity of biology:

This whole problem also got me thinking about my own subject area and my brief excursion into the study of restriction enzymes from Mycoplasma. Restriction systems are a means for bacteria to protect themselves from viral invasion and, despite the small size of the genomes of Mycoplasma bacterial strains, they encode such systems.  There is clear evidence that such systems are “selfish” and may be they are fundamental to the long term survival of bacteria, so I think they need to be dealt with in the model organism.  However, things begin to get complicated when you look at the well described system from Mycoplasma pulmonis (a slightly more complex version of the organism used for the model).  Instead of a single set of genes for a restriction system, as usually found in other organism, the restriction genes of Mycoplasma pulmonis  are capable of switching in a way that can generate four different proteins from a single gene.  This is where the complexity of modelling an organism occurs and while the organism used may have a simple genome, it is important to know of how even simple organisms can increase their genetic complexity without increasing their DNA content.

Conclusion:

I think the work at Stanford is both interesting and important and I think they have achieved a very important step along the road to modelling a living cell, but I also think they may need more information and have more complex modules available to them as they try to be more accurate with even these simple organisms.  It will be a long road before we have a model of a human cell, but what an incredible thought that would be!

 

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Synthetic Biology – will it work?

Eng_Future_Logo_OutlinesEvery now and then science comes up with a new approach to research that impacts on technology, but often these approaches are controversial and the headlines we see are far from the truth and can damage the investment into the new techniques.  One good example is the Genetic Modification of plants and the production of GM-foods, which has a really bad press in Europe despite many obvious benefits for the world economy and for disease control.  The latest technology, which follows from the explosion in genetic engineering techniques during the 1990s, builds on concepts developed in bionanotechnology and is known as Synthetic Biology.  But, what is Synthetic Biology?  Will it work?  And what are the dangers versus benefits of these developments?  Gardner and Hawkins (2013) have written a recent review about this subject, which made me think a blog on the subject was overdue.

My background in this area is two-fold:

  1. I was a part of a European Road-Mapping exercise, TESSY, that produced a description of what Synthetic Biology is and how it should be implemented/funded in Europe.
  2. I was also Project Coordinator for a European research project – BioNano Switch, funded by a scheme to support developments in Synthetic Biology, that aimed to produce a biosensor using an approach embedded in the concepts of Synthetic Biology.

So, what is Synthetic Biology?  I think the definition of this area of research needs to be clearly presented, something that was an important part of the TESSY project, as the term has become associated simply with the production of an artificial cell.  However, that is only one small aspect of the technology and the definition TESSY suggested is much broader:

Synthetic Biology aims to engineer and study biological systems that do not exist as such in nature, and use this approach for:

  • achieving better understanding of life processes,
  • generating and assembling functional modular components,
  • developing novel applications or processes.

syntheticBiologyThis is quite a wide definition and is best illustrated with a simple comparison – in electronic engineering there exists a blueprint (circuit diagram) that shows how components (resistors, capacitors etc.) can be fitted together in a guaranteed order to produce a guaranteed result (a device such as an amplifier).  The Synthetic Biology concept would be to have a collection of such components (DNA parts that include promoters, terminators, genes and control elements; cellular systems including artificial cells and genetically engineered bacteria capable of controlled gene expression; interfaces that can connect biological systems to the human interface for useful output).  This would mimic the electronic situation and provide a rapid mechanism for assembly of biological parts into useful devices in a reliable and predictable manner.  There are many examples of such concepts, but the best known is the Biobricks Foundadtion.  However, at the TESSY meeting I was keen to make it clear that there are fundamental problems with this concept, so what are the problems?

At its most simple concepts a Biobricks database would consists of a number of different types of DNA (promoters, are short DNA sequences that switch a gene on; terminators, are short DNA sequences that switch a gene off; control elements, are DNA sequences that control the promoter switching on or off a gene as required; genes, would be DNA sequences that produce Recombinant DNAbiotechnologically useful products; and cells, are the final package that enables the DNA to do its work and produce the required product), which sounds logical and quite simple.  However, biological systems are not as reliable as electronic systems and combinations of promoters and genes do not always work.  One of the major problems with protein production, using such artificial recombinant systems, is protein aggregation resulting in insoluble proteins that are non-functional.  In addition, there are many examples (usually unpublished) of combinations of Biobricks that do not work as expected, or if used in a different order also result in protein aggregation, none of which ever happens with electronic components.  The reasons are far from clear, but are closely related to the complexity of proteins and the need for them to operate in an aqueous environment.  My thoughts about how to deal with this situation is to have a large amount of metadata associated with any database of Biobricks, which includes information about failures or problems of protein production from specific combinations.  However, I am not aware of any such approach!

Synthetic CellThere are other aspects of Synthetic Biology that do not depend on Biobricks and one example is the artificial cell.  The ideal for such a system is a self-assembling package, capable of entrapping DNA, capable of replication and survival and able to produce useful biomaterials and significant steps have been made toward such a system.  However, one area of concern as such systems are developed, is containment – can we really be sure these artificial microbes will remain in a contained environment and not escape to interact with and possible change the natural bacterial population.  However, the power and capability of such a system should not be underestimated and the likely use in future medicine could be immense – simple examples would be as delivery systems for biomaterial that can activate cellular changes by targeting to the required cell and then switching on protein production (e.g. hormones).  This type of targeted medicine would be a major breakthrough during the later part of this century.

SEN25_BIO11Another type of Synthetic Biology involves the artificial assembly (possible self assembly) of biomaterials onto an artificial surface in an way that is unlikely to occur naturally, but provides a useful device – I see this as more like what a Biobricks project should be like – such a system is usually modular in nature and the bio-material would normally be produced using recombinant techniques.  The research project I mentioned earlier involved such a device and the outcome was a single molecule biosensor for detecting drug-target interactions at the limits of sensitivity.  The major issues we had with developing this device was the precise and accurate attachment of biomaterial, to a surface in such a way that they function normally.  However, overall the project was successful and shows that a Synthetic Biology approach has merits.

What are the benefits that Synthetic Biology can provide society?  Well, one advantage is a more systematic approach to biotechnology, which to date has tended to move forward at the whim of researchers in Academia or industry.  Assuming the problems, associated with protein production, mentioned above can be better understood then there could be a major boost in use of proteins for biotechnology.  In addition, Synthetic Biology techniques offer a unique opportunity for miniaturisation and mass production of biosensors that could massively improve medical diagnosis.  Finally, artificial cells have many future applications in medicine, if they can be produced in a reliable way and made to work as expected:

  1. They could provide insulin for diabetics.
  2. Be made to generate stem cell, which could be used in diseases such as Alzheimer’s and Huntingdon’s.
  3. They could deliver specific proteins, drugs and hormones to target locations.
  4. They could treat diseases that result from faulty enzyme production (e.g. Phenylketonuria).
  5. They could even be used to remove cholesterol from the blood stream.

However, there are always drawbacks and risks associated with any new scientific advance:switch%20off

  1. Containment of any artificial organism is the most obvious, but this enhanced by the possibility of using the organism to produce toxins that would allow its use as a biological weapon.
  2. The ability to follow a simple “circuit diagram” for protein production, combined with a readily available database of biological material, could enable a terrorist to design a lethal and unpredictable weapon much more complex and perhaps targeted than anything known to date.
  3. Inhibit research through a readily available collection of materials that prevent patent protection of inventions.  This could be complicated by the infringement of patents by foreign powers in a way that blocks conventional research investment.
  4. Problems associated with the introduction of novel nano-sized materials into the human body, including artificial cells, which may be toxic in the long term.

My own feeling is that we must provide rigorous containment and controls (many of which already exist), but allow Synthetic Biology to develop,  Perhaps there should be a review of the situation by the end of this decade, but I hope that the risks do not materialise and that society can benefit from this work.

High-level equipment and its impact on science

A recent article (Hamrang, et al. Trends in Biotechnology, 2013) made me think about the impact modern technology is having on how scientific research is developing and, in particular, my own experience of applying some of this technology.  I thought it might be interesting to detail some of this technology and how it has influenced my own research and how it might both develop to provide new approaches for the advancement of science and how this will change requirements in teaching.

AFM1A good place to start is SINGLE MOLECULE ANALYSIS a concept I had never thought of in my early research career, but it became a possibility during the 1990s.  The first time I  heard of single molecule analysis was something called a Scanning Tunnelling Microscope, but I could not see uses for this device outside of chemistry as the objects to be visualised were in a vacuum.  However, this device quickly developed into the Atomic Force Microscope (AFM) and the study of biological molecules was soon underway.  This device measures surface topology and can visualise large proteins as single molecules – my first involvement was to visualise DNA molecules translocationthat were being manipulated by a molecular motor.  The resolution was astounding, but more importantly we were able to use this technology to study intermediates that had been biochemically “frozen” in position and resolve features we never expected to see.  Further studies allowed us to also study protein-protein interactions and super-molecular assembly of the motor.  The wonderful thing about this technology is that interpretation of the data has quickly moved from the negativeness of “artefacts” and a lack of faith that images showed what was thought to be there, to a situation where major advancements are possible through direct topology studies.  Developments of this technology are likely to include automatic cell identification, in vivo measurements using fine capillary needles and measurements of ligand-surface target interactions on cells – this could influence drug development and biomedical measurements.  Another developing technology related to AFM is the multiple tip biosensor that can sense minute amounts of material in a variety of situations (a “molecular sniffer” – one use I heard of directly from the developer was for wine tasting/testing!
Magnetic TweezerMy second single molecule analysis involved a Magnetic Tweezer setup which is able to visualise movement of a magnetic bead attached to a single molecule (in our case DNA), which allowed us to determine how a molecular motor moves DNA through the bound complex, but, perhaps more importantly, this led us to develop a biosensor based around this technology that could be used to determine drug-target interactions at the single molecule level and perhaps allow single molecule sensing in anti-cancer drug discovery.  This technology is also closely related to optical tweezer systems that have been used in similar studies and the future is certain to make such technology cheaper and easier to use and their application in biomedical research.  The key to this development will be the increased sensitivity of single molecule studies and how this will enable more detailed understanding of intermediate steps in molecular motion induced by biomolecules. I imagine as newer versions of these devices become more automated, then they will be used as biosensors to study more complex systems that involve molecular motion.  In the short term, it seems to me that there is scope for the application of these devices in understanding protein amyloid formation and stability with a view to determining mechanisms for destabilising such structures.

SURFACE ATTACHED BIOMOLECULAR ANALYSIS.
SPRThe best known system in this category of analytical devices is Biacore’s Surface Plasmon Resonance (SPR), which uses a mass detection mechanism based on changes to the Plasmon effect produced by electrons in a thin layer of gold. We have used this to study protein-DNA interactions and subunit assembly and the technique provides a useful confirmation of older techniques such as electrophoresis. I have been involved in discussions about the application of this technology in the field, but reliability and setup problems remain a problem. In comparison, the Farfield dual beam interferometer can use homemade chips that simplify setup and seems more reliable for similar measurements. Where I see a potential for these devices is in the study of protein aggregation, which has tremendous potential in the study of amyloid-based diseases. This idea sprang from discussions with Farfield about using their interferometer to detect crystallization and would be an interesting project. However, if these devices are to have a major impact in biomedical sciences, they need to be easier to setup, more reliable and smaller.  recent advances are leading SPR toward single molecule sensing (Punj, D., et al., Nat Nano, 2013). I believe the real key to implementing this technology as a biosensor is to incorporate two technologies in the same device. We proposed to have a dynamic system, on an interferometer chip, whose activity would switch off the interferometre when active. This could be used in drug discovery, targeting the drug at two systems simultaneously. If massively parallel systems can be developed, possibly based around laminar-flow, I can see a use in molecular detection of hazardous molecules using either antibodies, or aptamers.

COMPUTER BASED IMAGE ANALYSIS.
cyroEMI have not directly used this technology, but I have seen the results applied to the molecular motor that I have worked with. The value of the system is that cyroEM allows the gathering of many images of a large protein complex, which allows structural studies of systems that cannot be crystallized or visualized using NMR. My feeling is that as computing power increases this technique combined with molecular modelling in silico, will provide structural information for many complex biological systems. The impact of this knowledge will greatly influence the design of drugs and will aid the biochemical analysis of complex systems. My feeling is that further development of this technology will revolve around combining it with other techniques for visualising biomolecules, one I have mentioned before is Raman Spectroscopy, which could allow studies of these complexes in situ another could be single molecule fluorescence (Grohmann, et al. Current Opinion in Chemical Biology). I can easily imagine collaborative research projects that will bring a variety of such techniques to the production of the 3D image of real biological systems isolated from cells. Such research would have to follow existing models of bidding to use such equipment in centres of excellence. Such centres would bring together visualization techniques with single molecule analysis and data from genomics and proteomics. The research lab of the future will depend on much more international collaboration than we have seen up to now!

STUDIES USING NANOPORES.
The current technology in this area divides into two types of nanopores, physical holes in a surface and reconstituted biological pores. I have used a physical nanopore to investigate the separation of proteins from DNA using electrophoresis across the nanopore, the beauty of this system is that it also quantifies the number of molecules crossing the pore. I imagine that such devices will develop using surface attached biomolecules around the pore, which will introduce specificity into the device, but what I would have liked to develop is a dynamic device for ordered assembly of molecules (an artificial ribosome) where the nanopore allow separation of the assembly line and the drive components – such are the dreams of a retired scientist!
nanopore_x616[1]Biological nanopores are the main focus for single molecule sequencing of DNA and the future must be portable, personal sequencing devices (DNA sequencing information must reside with the source of the DNA and for humans this will eventually lead to personal devices. However, the level of available data will be enormous and the growth of the “omics” research will require new ways to store, organise and access this information. A new method for studying biological systems is already underway in which analysis of data allows a better understanding of complex systems. This will eventually become a part of biomedicine and will be supported by personalised medicine.

I was once asked by a student what future Biology holds, and I now know it will be an area of significant growth for many years to come, but this requires the right focus for investment and a new direction for undergraduates in their studies – good luck to those I have taught, who now have to lead these developments.

Thoughts on single molecule science

Some while ago I worked with two groups who were using magnetic tweezers systems to study single molecule biophysics and I was always keen to expand that work to new projects F1_medium(unfortunately retirement moved such thoughts away from actuality).  A recent paper, using single molecule FRET analysis (Haller, A., et al. Proceedings of the National Academy of Sciences, 2013. 110(11): p. 4188-4193) has investigated the folding of an RNA aptamer.

This work encourages me to suggest further studies of aptamer folding using magnetic tweezer systems with a long-term view of developing a sensor for aptamer activity – it would be incredible to develop a toxin sensor, based on aptamers to specific toxins, that detects at the single molecule level!

Fig 10This is not such an over-ambitious project and relates closely to my own work, with a large European team, to develop a single molecule biosensor.  The basis of the three European projects, involved in this work, was to develop the magnetic tweezer device as an automatic electronic platform for detection of magnetic bead movement based on changes to the length of DNA molecules located within the device.  The concept was to use the device to identify drug-target interactions involving DNA-manipulating enzymes (such as helicases and topoisomerases) that are potential targets in anti-cancer drug development.

However, I always envisioned a greater potential for such a device and studying aptamers would be only one aspect of such a project.  The device could also be used to study Quadruplex formation, and drugs that affect this, other fibre formation, such as amyloids and even protein-protein interactions involving DNA binding enzymes to enable DNA-loop formation as an indicator of such interactions.  All of these uses provide a potential health-orientated development of the biosensor – just need someone to have the enthusiasm to write the proposal!

Could phage become useful viruses?

I remember many years ago my old professor telling me how bacteriophage could be useful tools for typing bacteria, based on their susceptibility to different restriction enzymes, which occur in different numbers in different bacteria, but, sadly, it never came to pass.  However, I read a recent paper (Henry & Debarbieux, 2012. Volume 434, 151–161) that suggest a new set of tools could be made available for healthcare that could make use of viruses and in particular bacteriophage.

bacteriophageSo, what are bacteriophage?  Well these are viruses that specifically infect bacteria and there thousands of them for all of the different bacteria known (and probably many more as yet unknown) and often when they do infect and kill the host, they use the bacterial replication system to copy themselves many times.  As rather beautifully illustrated on the left, they attach to the surface of a bacteria, using a highly specific attachment mechanism, and then inject their nucleic acid (DNA or RNA) into the cell like a biological hypodermic needle.  If grown artificially, in a laboratory, on a surface coated with growing bacteria, this produces areas of killing know as plaques – an example is illustrated at http://www.typei-rm.info/background.htm – the idea of typing bacteria is based on changes to the number of plaques by the defence systems bacteria use to stop phage growth – restriction enzymes.  Each strain of bacteria carries a different array of restriction enzymes, which should produce different efficiencies of growth of saturating concentrations of phage, sadly, the restriction enzymes are so efficient at killing the phage that growth show little variation between the many strains!

Since my early days in molecular biology, bacteriophage have been manipulated and the genomes have been deleted or changed to make use of them as the tools of molecular biology.  In fact, it is really the success of work by Prof Noreen Murray that initiated the ideas of using viruses as molecular tools; she was able to produce altered bacteriophage genomes that now carried “foreign” DNA and this produce some of the first genetically engineered organisms.

CaptureViruses are already used to deliver both drugs and to engineer genomes against disease, but how this might work and how useful viral tools might be best illustrated by a mechanism know as Phage Display, which has been used to produce artificial antibodies without recourse to injection of mice or other mammals!  The antibody proteins are attached to the outside of the bacteriophage, where they are functional, and they can be used to bind to an antigen attached to a surface (for example an antigen marker that is known to occur in cancer cells), the phage that bind can be isolated, manipulated genetically to improve binding to the antigen and then purified under more stringent procedures – panning.  Eventually, an antibody is isolated that is a tight binder to the antigen and could be used therapeutically.  However, one can imagine the next step in this type of work would be to target viruses at cancer cells, using viral display of specific antibodies, and then kill the cancer cells using part of the viruses normal life cycle.

So, do viruses hold a useful future for genetic engineers and bionanotechnologists?  I would say, as we begin to fully understand the genetics of the many viruses and learn to manipulate the genomes in a safe way, we could very well have a series of tools for manipulating genetic information, or for that detection, diagnoses, control and even cure of infectious diseases.  Who knows whether in another decade, during which time many new viruses will be discovered, the possibilities are enormous!

My research finally concludes…..

Tomorrow sees the viva for my last ever PhD student; although, I only ever supervised him for his first year.  I wish you well Luke and I am sure you will be fine.

However, as the title of this Blog indicates this really draws my research career to an end, which seems somewhat strange after all of these years.  I started working in the area of Type I Restriction-Modifcation systems way back in 1974, when I moved from Hull back to my birthplace – Newcastle upon Tyne – to work for Prof Stuart Glover.  But my own research really started when I moved to Portsmouth in 1988 (almost 25 years ago).  The direction changed gradually from the biochemistry of these enzymes, how they manipulate DNA and control the opposing activities of restriction and modification through to direct visualisation of active single molecules using Atomic Force Microscopy.

But, introducing these enzymes into a single molecule biosensing device was the most unexpected outcome of the work, which led me down a path toward nanotechnology and possible commercialisation – a long way from a study of something once described as “esoteric enzymes”.

Retirement is a pleasure and I don’t miss the stress of research, but I do miss the challenge of writing successful grant applications (along with all the required unsuccessful ones, lol).  My final contribution is a book describing Molecular Motors and their possible uses in Bionanotechnology.

Scientific Research Paper of the month – a single molecule car!

A recent paper in nature describes a single molecule machine capable of directed motion along a surface (Kudernac, et al. (2011). Electrically driven directional motion of a four-wheeled molecule on a metal surface. Nature 479, 208-211).

One of the major challenges of Nanotechnology is to provide a mechanism for moving single molecules in a directed manner so that they might be assembled in highly organised ways. One mechanism is to use the inherent ability of certain molecules to self-organise or self-assemble and this can be very successful for assembly of large arrays of the same molecule. Perhaps the best example of the use of self-assembly is provided by Nature’s machines and is well illustrated by the bacterial flagella, which self assembles from the constituent proteins.

AFM1However, self assembling systems are difficult to control and a more controlled situation is where single molecules are transported from one place to another. To date this has been achieved by manipulating single molecules using an Atomic Force Microscope , which can physically lift a molecule off a surface and drop the molecule elsewhere. but an ultimate ideal would be a machine that could carry molecules around a surface.  Nature accomplishes this using a molecular motor – kinesin, but this system requires a “track” for the motor to run along.

The motor described in this Nature paper is composed of four rotary chemical motors, which are linked together by a central axis.  The motors are driven by electron tunnelling, which drives the machine across a copper surface.  Direction is changed by specific activation of the individual motors  This machine is a first step toward a device capable of independent motion, with the ability to carry cargo.  The chemical nature of the motors makes attachment of other molecules a simple process and the required drive force could be produced from a variety of sources – light, chemical and electrical.

Watch this space for future developments!