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Biofuel Production and Synthetic Biology
Bringing the costs of producing these advanced biofuels down to competitive levels
with petrofuels, however, is a major challenge. Researchers at the U.S. Department
of Energy (DOE)s Joint BioEnergy Institute
(JBEI), a bioenergy research center led by
Berkeley Lab, have taken another step towards meeting this challenge with the development of a new technique for pre-treating cellulosic biomass with ionic liquids
- salts that are liquids rather than crystals at
room temperature. This new technique requires none of the expensive enzymes used
in previous ionic liquid pretreatments, and
makes it easier to recover fuel sugars and
recycle the ionic liquid.
Most of our ionic liquid efforts at JBEI have
focused on using enzymes to liberate fermentable sugars from lignocellulosic biomass after pretreatment, but with this new
enzyme-free approach we use an acid as
the catalyst for hydrolyzing biomass polysaccharides into a solution containing fermentable sugars, says Blake Simmons, a
chemical engineer who heads JBEIs Deconstruction Division and was the leader of
this research. Were then able to separate
the pretreatment solution into two phases, a
sugarrich
water phase for recovery and a lignin-rich
ionic liquid phase for recycling. As an addedbonus, our new pretreatment technique
uses a lot less water than previous pretreatments.
With the burning of fossil fuels continuing to
add 9 billion metric tons of excess carbon
dioxide to the atmosphere each year, the
need for carbon neutral, cost-competitive
renewable alternative fuels has never been
greater. Advanced biofuels, produced from
the microbial fermentations of sugars in lignocellulosic biomass, could displace gasoline, diesel and jet fuel on a gallon-for-gallon
basis and be directly dropped into todays
engines and infrastructures without impacting performance. If done correctly, the use
of advanced biofuels would not add excess
carbon to the atmosphere. Environmentally benign ionic liquids are used as green
chemistry substitutes for volatile organic
solvents. While showing great potential as a
biomass pretreatment for dissolving
lignocellulose and helping to hydrolyze the
resulting aqueous solution into fuel sugars,
the best of these ionic liquids so far have
required the use of expensive enzymes.
Recent studies have shown that acid catalysts, such as hydrochloric or Brnsted, can
effectively replace enzyme-based hydrolysis, but the subsequent separation of sugars and ionic liquids becomes a difficult and

expensive problem can require the use of


significant amounts of water.
Guided by molecular dynamics simulations carried out at DOEs National Energy Research Scientific Computing Center
(NERSC), Simmons and his colleagues at
JBEI solved this problem by deploying the
ionic liquid imidazolium chloride in tandem
with an acid catalyst as Imidazolium is the
most effective known ionic liquid for breaking down lignocellulose and the chloride
anion is amenable with the acid catalyst.
The combination makes it easy to extract
fermentable sugars that have been liberated from biomass and also easy to recover
the ionic liquid for recycling. By eliminating
the need for enzymes and decreasing the
water consumption requirements of more
traditional ionic liquid pretreatments we
should be able to reduce the costs of sugar production from lignocellulose. Complete
separation of the pretreatment solution into
sugar-rich water and lignin-rich ionic liquid
phases was attained with the addition to the
solution of sodium hydroxide.
The production of commercially attractive
biofuels using enzymatic methods, all the
same, is not as easy as it appears. The various polysaccharides viz. cellulose, starch,
lignin, hemicellulose, or lignocelluloses
need to be enzymatically degraded for their
transformation into glucose or sugar molecules which in turn are fermented into biofuels (bioethanol or biobutanol). In case of cellulose, the process of cellulolysis involves
enzymes like cellulases and glucosidases.
Cellulases are expensive, unstable and slow
in action; therefore they increase the overall
economics of the process of cellulolysis and
hence biofuel production. The bulk production of cellulases at industrial level seems
to be the relevant solution. The microbes
that produce cellulases include symbiotic
anaerobic bacteria (e.g. Cellulomonasfimi,
Clostridium thermocellum, Clostridium phytofermentans, Thermobifidafusca ) found in
ruminants such as
cow and sheep, flagellate protozoa present
in hindguts of termites, and filamentous fungi isolated from decaying plants (e.g. Hypocreajecorina,
Thermoascusaurantiacus,
Phanerochaetechrysosporium, Neurosporacrassa, Tricodermareesei, Asperigillusniger, Fusariumoxysporum). The gene(s)
responsible for cellulase production are
characterized, isolated and recombinantly
introduced into Escherichia coli for the enhancedcellulase expression levels. Apart
from the conventional biotechnology methods for biofuel production, synthetic biology
has shown promising results lately.

Understanding the DNA sequences, precisely measuring the gene behaviour paves
way for fabricating or synthesizing the cellulase gene de novo. To put it in simple words,
synthetic biology is a science of designing
and constructing new biological parts, devices and systems for programming cells
and organisms and endowing them with
novel functions. It is a technique of writing
the DNA / genetic code base by base using
several computational tools and softwares
like Gene designer, GenoCAD, Eugene and
Athena to name a few. Gene designer is a
DNA design tool for de novo assembly of
genetic constructs, GenoCAD is a computer-assisted-design application for synthetic
biology for designing complex gene constructs and artificial gene networks, Eugene
is a language designed to develop novel
biological devices and Athena is a CAD /
CAM software for constructing biological
models asmodules. These synthetic biology
approaches can be useful in bringing down
the cost of cellulases and, thereby, of biofuels. Several companies are spending a fortune on the production of bioethanol for example; Amyris Biotechnologies, Verenium,
Iogen, Bioethanol Japan, Mascoma, POET,
SolixBiofuels, Pacific Ethanol, NextGen
Fuel Inc. and Jatro Diesel. However, the
cost-effective production of the second generation biofuels is still a cherished desire of
the scientific community. Synthetic biology
is an evolving field still dealing with the inherent complexity of biological systems and
overcoming the biosafety issues involved
with engineering the living systems. Indeed
the proliferation of the computer modelling
tools is leading to the revolution of this discipline which might write the success story
of some of the present and future scientific
challenges.

Biodegradable Battery: The battery that melts inside the body!


A four-cell battery made of biodegradable artery stent that has been successfully
materials completely dissolves after three tested in clinical trials, and a concentraweeks in water.
tion that is unlikely to cause problems in
the body, says Rogers. Almost all of the
Such biodegradable, implantable battery key building blocks are now available to
could help in the development of biomedical produce self-powered, biodegradable imdevices that monitor tissue or deliver treat- plants, he says.
ments before being reabsorbed by the body
after use. This is a really major advance, All versions can maintain a steady output
says Jeffrey Borenstein, a biomedical en- for more than a day, but not much longer.
gineer at Draper Laboratory, a non-profit The team hopes to improve the batteries
research and development center in Cam- power per unit weight known as powbridge, Massachusetts. Until recently, there er density by patterning the surface of
has not been a lot of progress in this area. the magnesium foil to increase its surface
In 2012, materials scientist John Rogers at area, which should enhance its reactivity.
the University of Illinois at Urbana-Cham- The authors estimate that a battery meapaign unveiled a range of biodegradable sil- suring 0.25 cm2 and just one micrometer
icon chips that could monitor temperature thick could realistically power a wireless
or mechanical strain, radio the results to implantable sensor for a day.
external devices, and even heat up tissue
to prevent infection. Some of those chips re- In the field
lied on induction coils to draw wireless pow- The devices could also find environmener from an external source.
tal applications, says Borenstein. For exBut wireless power transfer is problemat- ample, to help remediation efforts during
ic for devices that need to go deep within an oil spill, environmental officials could
tissue or under bone. The components that drop hundreds of thousands of tiny wirereceive the power are also quite complex, less chemical sensors across the slick.
anything put in is going to take space.. To These would later simply dissolve in
provide a tidier solution, Rogers and his col- the ocean. Space is less of a constraint
laborators have now created a fully biode- in these applications: a stack of several
gradable battery.
cells, for instance, can produce up to 1.6
volts enough to power a light-emitting
Dissolvable devices
diode or generate a radio signal.
These devices use anodes of magnesium
foil and cathodes of iron, molybdenum or Magnesium batteries are not the only
tungsten. All these metals slowly dissolve in solution. Last year, biomaterials scientist
the body, and their ions are biocompatible in Christopher Bettinger of Carnegie Mellon
low concentrations.
University in Pittsburgh, Pennsylvania,
The electrolyte between the two electrodes unveiled an edible sodium-ion battery
is a phosphate-buffered saline solution, with electrodes made from melanin pigand the whole system is packed up in a ments. But Rogers team report that their
biodegradable polymer known as a poly- magnesium batteries have a relatively
anhydride. Currents and voltages vary de- higher current and power density, and
pending on the metal used in the cathode. last for longer.
A one-square-centimeter cell with a 50-micrometer-thick magnesium anode and an Borenstein hopes that further research
8-micrometer-thick molybdenum cathode
into both types of batteries could evenproduces a steady 2.4 milliamps of current, tually yield implantable drug-delivery defor example.
vices that are controlled by radio signals,
or that dispense pharmaceuticals in reOnce dissolved, the battery releases less sponse to a specific acute problem, such
than 9 milligrams of magnesium roughly as an epileptic seizure.
twice as much as a magnesium coronary

Light Activated Neurons From Stem Cells Restore


Functions to Paralyzed Muscles

The Concept:
A new way to artificially control muscles using light, with the potential to restore function to muscles paralyzed by conditions
such as motor neuron disease and spinal
cord injury, has been developed by scientists. The technique involves transplanting
specially designed motor neurons created
from stem cells into injured nerve branches.
These motor neurons are designed to react
to pulses of blue light, allowing scientists to
finetune muscle control by adjusting the intensity, duration and frequency of the light
pulses.

The Technique:
The technique involves transplanting specially designed motor neurons created
from stem cells into injured nerve branches. These motor neurons are designed to
react to pulses of blue light, allowing scientists to finetune muscle control by adjusting
the intensity, duration and frequency of the
light pulses.
Following the new procedure, we saw previously paralyzed leg muscles start to function, says Professor Linda Greensmith of
the MRC Centre for Neuromuscular Diseases at UCLs Institute of Neurology, who
coled the study. This strategy has significant advantages over existing techniques
that use electricity to stimulate nerves,
which can be painful and often results in
rapid muscle fatigue. Moreover, if the existing motor neurons are lost due to injury or
disease, electrical stimulation of nerves is
rendered useless as these too are lost.
Muscles are normally controlled by motor
neurons, specialized nerve cells within the
brain and spinal cord. These neurons relay
signals from the brain to muscles to bring
about motor functions such as walking,
standing and even breathing. However, motor neurons can become damaged in motor neuron disease or following spinal cord

injuries, causing permanent loss of muscle


function resulting in paralysis.
This new technique represents a means
to restore the function of specific muscles
following paralysing neurological injuries or
disease, explains Professor Greensmith.
Within the next five years or so, we hope to
undertake the steps that are necessary to
take this groundbreaking approach into human trials, potentially to develop treatments
for patients with motor neuron disease,
many of whom eventually lose the ability to
breathe, as their diaphragm muscles gradually become paralyzed. We eventually hope
to use our method to create a sort of optical
pacemaker for the diaphragm to keep these
patients breathing.
We custom-tailored embryonic stem cells
so that motor neurons derived from them
can function as part of the muscle pacemaker device. says Dr Lieberam, who coled
the study. First, we equipped the cells with
a molecular light sensor. This enables us to
control motor neurons with blue light flashes. We then built a survival gene into them,
which helps the stemcell motor neurons to
stay alive when they are transplanted inside
the injured nerve and allows them to grow
to connect to muscle.

Self healing engineered muscles grown in laboratory


The Concept:
Living skeletal muscle that contracts powerfully and rapidly, integrates quickly into
mice, and for the first time, demonstrates
the ability to heal itself both inside the laboratory and inside an animal has been
grown in the lab by biomedical engineers.
The muscle we have made represents an
important advance for the field, an author
said. Its the first time engineered muscle
has been created that contracts as strongly
as native neonatal skeletal muscle.
The Technique:
The study conducted at Duke University
tested the bioengineered muscle by literally
watching it through a window on the back of
living mouse. The novel technique allowed
for realtime monitoring of the muscles integration and maturation inside a living,
walking animal. Both the labgrown muscle
and experimental techniques are important steps toward growing viable muscle for
studying diseases and treating injuries, said
Nenad Bursac, associate professor of Biomedical Engineering at Duke. Every muscle
has satellite cells on reserve, ready to activate upon injury and begin the
regeneration process. The key to the teams
success was successfully creating the microenvironments called niches where these

stem cells await their call to duty. Simply


implanting satellite cells or lessdeveloped
muscle doesnt work as well, said Juhas.
The well-developed muscle we made provides niches for satellite cells to live in, and,
when needed, to restore the robust musculature and its function.
How the trials took place:
To put their muscle to the test, the engineers
ran it through a gauntlet of trials in the laboratory. By stimulating it with electric pulses, they measured its contractile strength,
showing that it was more than 10 times stronger than any previous engineered muscles.
They damaged it with a toxin found in snake
venom to prove that the satellite cells could
activate, multiply and successfully heal the
injured muscle fibers.
Then they moved it out of a dish and into
a mouse. With the help of Greg Palmer, an
assistant professor of Radiation Oncology
in the Duke University School of Medicine,
the team inserted their labgrown muscle
into a small chamber placed on the backs
of live mice. The chamber was then covered
by a glass panel. Every two days for two
weeks, Juhas imaged the implanted muscles through the window to check on their
progress.

By genetically modifying the muscle fibers


to produce fluorescent flashes during calcium spikes which cause muscle to contract
the researchers could watch the flashes become brighter as the muscle grew stronger.
We could see and measure in real time
how blood vessels grew into the implanted
muscle fibers, maturing toward equaling the
strength of its native counterpart, said Juhas.
The engineers are now beginning work to
see if their biomimetic muscle can be used
to repair actual muscle injuries and disease.
Can it vascularize, innervate and repair the
damaged muscles function? asked Bursac.
That is what we will be working on for the
next several years.

focus

I
N

optogenetics
see it, without actually seeing it

When you think of the brain what


do you see; a group of identical
neurons packed together forming different regions of the brain
right?? Well you are wrong, there
are many distinct types of neurons,
which are distributed in a highly
organised fashion in different brain
regions and interconnected with
exquisite specificity. And when I
say many, I mean many hundreds,
possibly thousands of types.
Now imagine a way you could excite a single type, just to see its effect on the organism in real time,
welcome to the world of OPTOGENETICS.
Introduction:
Optogentics is a new method of
neural modulation that can even be
applied to freely moving animals.
Strictly speaking, optogenetics involves experimenting with a combination of genetic manipulation
and optics It can be used in several animal models, including the
C. elegans, fly, zebrafish, mouse,
rat, and primate .Ultimately, optogenetics may be used to control the behavior of freely moving
mammals by administering light. A
complicated explanation wouldnt

you say, so lets break it down.

ic cells instead. For this to happen


we need to use various methods
of recombinant DNA technology to
facilitate said transfer of genes.
One possibility is to use a vector,
for example inject a (harmless)
virus to carry the opsin gene into
the brain of a mammal. The major
drawback of viral expression systems is that they cannot carry large
amounts of genetic material. However, the advantage is that opsins
are expressed in high levels. An-

The first key component of this


technology (I use technology in
the more liberal sense) are opsin
, these are light sensitive G-coupled proteins ( transmemberane
proteins that are capable of converting a stimulus into a cellular response) . There are broadly of two
types Type I opsins are employed
by prokaryotes, whereas animals
use Type II opsins. Each opsin type
reacts to a certain wavelength in a
unique manner. This allows them
to be used for not just activation
but for inhibition as well. Now cell
with these
proteins react by opening a channel in their cell membrane to allow
electrochemical ions (like sodium or chloride) to flow in or out of
Use of programmable LCD photomask
for neuron stimulation
the cell when they are subjected
to light of a light of certain wavelengths. Controlling the flow of other way to introduce opsins is to
such ions along their fibres is also use transgenic (knock-in) animals
how neurons conduct electricity.
that possess the opsins from birth.
This obviously has the advantage
The next step is to make sure that of studying the development of a
mammalian cells express microbi- system. However, transgenic anial opsins. Because simply admin- mals show lower opsin expression
istering a protein will not work, a level. Other methods of transfer ingene that encodes for the opsin clude the use of Cre-Lox recombineeds to be introduced to specif- nase system, a widely used meth-

Schematic diagram of
Light-based stimulation

od to make changes into the DNA


at specific locations. Now if you
were to stimulate a particular site
in the brain of the animal you
would see that only a specific type
of neurons that express the opsin
fire, ie are excited or activated or
are inhibited. This depends on the
type of opsin used. For example
blue light makes channelrhodopsin-2 (ChR2) rapidly depolarize a
neuron.
Now why would we go through
all this trouble? I am sure you are
wondering whether there are any
existing ways of checking neural
response, well there are but many
are nowhere near as specific and
some are downright weird (for example deep brain stimulation involves placing a device in the brain
which sends electrical impulses to
specific parts of the brain, it is used
for treating diseases like Parkinson`s )optogenetics provides one
means to integrate analyses at
very different levels, uniting what
have been disparate areas of neuroscience.
The characteristics of individual
neurons or specific synaptic connections are traditionally analysed
by molecular and cellular neuroscience and electrophysiology.

with pharmacology. The functions


and interactions of brain areas
are studied using field recordings,
electroencephalography, neuroimaging, lesions and other systems
neuroscience methods.
The advantage of optogenetics over other neuromodulation
techniques is its high-temporal
specificity combined with cellular
precision. For example, although
electrical manipulation has a
high temporal resolution (precise
time dependent measurements),
it is unable to achieve true inactivation or excitation of individual neurons. Pharmacological and
genetic manipulations show the
opposite pattern, they can target
at least certain kinds or families of
neurons but are lacking temporal
precision. Thus by allowing us to
better understand the functioning
of various neuron types, their interactions with each other and the
effect of pharmacological agents
on them; optogenetics gives us a
far more holistic approach to the
study of the brain that too is a very
efficient manner.

Optogenetics as an important
research tool:
Relationship between dopamine
(DA) neuron firing and positive
reinforcement in genetically modified rats. The use of optogenetics
can lead to a better understanding
of cause-effect relationships, for
example in dopamine-based disorders such as Parkinson`s.
Understanding of sleep patterns.
Optogenetics is important for controlling neural circuits to examine
boundaries between sleep and
The roles of specific neurotrans- wakefulness.
mitters or receptors are probed Several studies used optogenet-

ics to investigate neural circuits


that underlie fear conditioning and
memory formation, these play an
important role in theunderstanding
of fundamental cognitive processes like memory formation, but also
in anxiety disorders and post-traumatic stress disorder, which are
characterized by disturbing, recurring contextual memories.
Optogenetics has further been applied to cortical oscillations (synchronized neural activity), that are
associated with various cognitive
processes as well as psychiatric
conditions such as anxiety, autism,
and schizophrenia.
Conclusion:
In all Optogenetics provides us a
tool to study a myriad set of diseases and disorders, with a level
of specificity that was previously
not possible to achieve. It gives us
the chance to combat diseases
and disorders that were previously
thought untreatable, not to mention greater insight into the functioning of the most mysterious and
important organ of our bodies.
Some of the most important work
in the field was done in 2005, by
researchers at Karl Deisseroths
laboratory first demonstrated a
single-component
optogenetic
system and in 2006 the term optogenetics was born.
Research in this field is being carried out in many of the top universities in the world. For instance,
optogenetic modulation in primate
neurons has been investigated and
it has already been demonstrated
that ChR2 can function within human neurons.

EDITORS

Kanwar abhay singh

Creative Team

Ashima Aggarwal

gagan bhasin

Ujjal Didar Singh

Pujit Singh