What we show in our project is only the tip of thewhole iceberg. More possible applications of halorhodopin could benefit notonly synthetic biology but also the human society. Here we propose several moreadvanced applications of halorhodopsin.

Halorhodopsin could facilitate light detection andlight-coupled inter-cell signal transduction. Light signal could be convertedto intracellular chloride level signal, which thus induces P_gaddownstream genes. The target genes could be constructed to accelerate thesynthesis pathway of quorum sensing signals, such as N-acyl homoserine lactones(AHL)¹. As a result,one clone of bacteria could be designed as light detector, amplifying andconverting light signal to quorum signal to cooperate with other clones ofbacteria.

Moreover, halorhodopsin is not the only channel thatcouples light to pump ions. There are other ion channels from retinylideneprotein family sharing similar sequences and structures but pumping other ionsusing light, such as bacteriorhodopsin pumping proton ions out of cells² andchannelrhodopsin non-specifically pumping cations into cells³. Combining pHsensitive promoter⁴ with bacteriorhodopsinand osmolality sensitive promoters (OmpF, developed by iGEM08_NYMU-Taipei in2008) with channelrhodopsin,   the light-coupled expression platform can beextended to more accurate and more complex regulation, which facilitatesbacteria being more programmable by computer.

Together with channelrhodopsin, halorhodopsin alsoenables water desalination utilizing solar energy. Sea water desalination haslong been attractive and difficult to implement efficiently. E. coli which can express halorhodopsinand channelrhodopsin could be one promising way to solve this problem. It ispossible to drive E. coli to capturevarious ions (mainly sodium ions, potassium ions and chloride ions) in seawater and move these ions away by controlling the movement of E. coli. By this means sea water couldbe desalinated for drinking, irrigation or other daily usage.

In conclusion, halorhodopsin could fulfill the needof utilizing light as signal or energy resource. We believe that halorhodopsinwould be one of the most interesting tools of synthetic biology in the future.

 

References

1.        Shapiro, J. a Thinking about bacterialpopulations as multicellular organisms. Annual review of microbiology 52,81-104(1998).

2.        Hayashi, S., Tajkhorshid, E. & Schulten, K. Moleculardynamics simulation of bacteriorhodopsin’s photoisomerization using ab initioforces for the excited chromophore. Biophysical journal 85,1440-9(2003).

3.        Nagel, G. et al. Channelrhodopsin-2, a directly light-gatedcation-selective membrane channel. Proceedings of the National Academy of Sciences100, 13940(2003).

4.        San, K. et al. An Optimization Study of a pH-InduciblePromoter System for High-Level Recombinant Protein Production in Escherichiacoli. Annals of the New York Academy of Sciences 721,268–276(1994).

 

 

Project Overview

 

Halorhodopsin, a light-driven ion pump originated from Halobacterium, employs light to transport chloride ions into cells uni-directionally against osmolality. Since chloride ion is a ubiquitous and essential element in most biological systems, halorhodopsin has thefascinated property to transform solar energy, the most abundant energy in theworld, into intracellular chloride ion level. However, this specificity of halorhodopsinhasn’t been well regulated and characterized in the previous competition. It is of our great interest to characterize its properties, and eventually utilize them in our project, seeking the possibilities tobenefit synthetic biology and the human society.

 

In our project, we integrated halorhodopsininto functional biobricks. Based on the characterized properties ofhalorhodopsin, we have furthermore developed and implemented two innovativeapplications: Light-coupled computer-aided expression platform and entropy-mixingelectrical power generation. The exciting results show us the power and thepotential of this fascinating tool. We are glad to introduce our achievements inthe project session.

 

Besides the proof-of-conceptdesigns in our projects, any design with the linkage of light and chloride ionscan be accomplished by this tool. Thus, there are much more potentialapplications development. We believe that halorhodopsin would be an excitingand useful biobrick in the future.

Field: Project

Block: Background

Session: P_gad introduction

 

P_gad is chloride-sensitive promoter whichwas first discovered in Lactococcuslactis¹, whichis a gram-positive bacterium which canlive in acidic environment. P_gad operon (Fig. 1) provideshydrochloric acid feedback mechanism to adjust intracellular metabolism, inorder to survive in acidic environment². In thisoperon, gadC is glutamate-gamma-aminobutyrate antiporter and gadB is glutamatedecarboxylase. They are both involved in intracellular pH regulation andco-expressed in the same operon under the control of P_gad². The genebefore P_gad, named gadR, which is constitutively expressed under thecontrol of P_gadR, is a positive regulator of P_gad coupledgenes while intracellular chloride is level elevated². Whenintracellular pH decreases, the expression of gadB and gadC is enhanced due tothe action of gadR and confers glutamate-dependent acid resistance in L. lactis².

J. Sanders et al. tried to developchloride-sensitive expression cassette using P_gad operon³.  They constructed the cassette from bp 821 to2071 of GenBank sequence AF005098, which includes P_gadR, gadR, P_gadand the starting codon ATG, and replaced downstream report genes³. They managetransforming the cassette to E. coliand varying the expression of report genes under different sodium chlorideconcentrations³. In ourproject, we try to build light-coupled chloride expression switch based on thisdesign.

References

1.        Sanders, J.W. et al. Identifcation of asodium chloride-regulated promoter in Lactococcus lactis by single-copychromosomal fusion with a reporter gene. Mol Gen Genet 257, 681-685(1998).

2.        Sanders, J.W. et al. A chloride-inducible acid resistancemechanism in Lactococcus lactis and its regulation. Molecular microbiology27, 299-310(1998).

3.        Sanders, J.W., Venema, G. & Kok, J. A chloride-induciblegene expression cassette and its use in induced lysis of Lactococcus lactis. Appliedand environmental microbiology 63, 4877(1997).

 

 

 

Electricity generation

From salinity to electricity

A novel nano-electrode has been proposed by Fablo. et. el that electrical energy can be generated by alternating salinity difference. With the materials sponsored from companies in mainland China in surprisingly large quantities, we have reproduced a even larger electrode (in surface area) with ease. With this at hand, we can turn salinity difference from bacterial action to electrical energy we can use.

The graph above showed the voltage variation of our cell against time.

From the graph above, it is clear that the voltage generated from the cell culture cannot power up any daily electronic device.

Power accumulation

An IC manufactured by Seiko, S-882Z, is a voltage booster that accepts input voltage down to 0.3V. This IC is produced through fully-depleted Silicon-On-Insulator technology that enables such low voltage input. The output is 1.8V/100uA; this voltage is used to charge up a super-capacitor. The supercapacitor can act as a voltage source for dc-dc converters, provide up to 5V for low-power device applications.

 Previousrelated projects

 

In 2010 iGEMcompetition, Queens-Canada team submited halorhodopsin from H. salinarum as biobricks and inserted thisgene to C. elegans. However, it wasnot well characterized. This year, we are trying to clone halorhdopsin from N. pharaonis, which has already beensuccessfully introduced and proved to perform complete light cycles in E. coli, to our biobrick system¹. We aim to characterize the efficiency ofthis halorhodopsin to be a well-documented biobrick and a useful tool in E. coli.

 

In previous iGEMprojects, various light sensors have been developed, including red light sensor(UT Austin, 2004), green light sensor (Tokyo-Nokogen, 2009) and blue lightsensor (University of Edinburgh, 2010). They are all light-induced fusiontranscription factors that trigger gene expression under the control ofspecific promoters, facilitating simply on/off switch and light-coupledcommunication. However, our design makes halorhodopsin not only a dynamic tunablelight sensor – by coupling with chloride sensitive promoters (e.g. P_gad),but also an energy converter – by storing solar energy as osmolality potentialand further converted to electricity. Our project would provide a wilder scopeof applications from signal transduction and gene regulation to energygeneration.

 

 

References

1.        Hohenfeld, I. Purification of histidinetagged bacteriorhodopsin, pharaonis halorhodopsin and pharaonis sensoryrhodopsin II functionally expressed in Escherichia coli. FEBS Letters 442,198-202(1999).

 

 

 

Entropy-mixingbattery

 

1.     Introductionof mechanism

In the nature, the water cycle is drivenby solar energy. One part of the water cycle is that solar energy evaporateswater in the sea to become fresh water through inland precipitation. Fromanother point of view, during evaporation, the entropy of different ions in thesea, mainly sodium, chloride and potassium, decreases as ion concentration iselevated. It is a common phenomenon when high concentration solution of acertain solvent, such as sodium chloride, is diluted, the entropy of thesolvent increases and the energy is released as heat. Thus the ocean isactually a gigantic energy reservoir. Its energy is transformed from solarenergy and stored as salinity potential. When sea water mixes with fresh waterfrom river, massive amount of energy is released.

Fig.1  Cycles of mixing-entropy battery.

 

 

 

Video is here

 

Recently, some institutes are devotinggreat efforts to seek efficient methods of extracting energy released frommixing sea water and fresh water. Mixing-entropy battery is thus designed toconvert salinity potential to electricity¹(Fig. 1). One pair of electrodes can specifically bind sodium ions or chlorideions, thus separating the charge when they are immersed in high salinitysolution, while decreasing the sodium chloride concentration in the solution.During this process, electrons in the cathode flow across electrical wires andreach the anode when there is complete electric circuit, since the immobilenegative charges (chloride) accumulating in the cathode repels electrons, whileimmobile positive charges (sodium) accumulating in the anode attract electrons.When the electrodes achieve equilibrium with the solution, there is no electriccurrent anymore. The next step is to immerse full-loaded electrodes in freshwater. Due to the salinity difference, sodium ions and chloride ions arereleased from the electrodes and those exceeded electrons in anode flow back tocathode to resume the original state. When the equilibrium is achieved, theelectrodes are re-immersed to high salinity water to start another cycle¹.

 

However thismethod only has high efficiency near estuaries. To solve this limitation, were-design this method using halorhodopsin-transformed E. coli. In our project, we fabricated the pair of electrodesaccording to W. Guo’s method² and withdrewcurrents from the battery. This is the first attempt to generate electricityfrom light energy by microorganism system in iGEM competition.

 

 

 

 

References

1.        La Mantia, F. et al.Batteries for Efficient Energy Extraction from a Water Salinity Difference. Nanoletters 0-3(2011).at <http://pubs.acs.org/doi/abs/10.1021/nl200500s>

2.        Guo, W. et al. Energy Harvesting withSingle-Ion-Selective Nanopores: A Concentration-Gradient-Driven NanofluidicPower Source. Advanced Functional Materials 20, 1339-1344(2010).