Team:Hong Kong-CUHK/Project/background

Background

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 glutamate decarboxylase. 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 develop chloride-sensitive expression cassette using P_gad operon³.  They constructed the cassette from bp 821 to 2071 of GenBank sequence AF005098, which includes P_gadR, gadR, P _gadand the starting codon ATG, and replaced downstream report genes³. They manage transforming 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).

Previous related projects

In 2010 iGEM competition, Queens-Canada team submited halorhodopsin from H. salinarum as biobricks and inserted this gene to C. elegans. However, it was not well characterized. This year, we are trying to clone halorhdopsin from N. pharaonis, which has already been successfully introduced and proved to perform complete light cycles in E. coli, to our biobrick system¹. We aim to characterize the efficiency of this halorhodopsin to be a well-documented biobrick and a useful tool in E. coli.

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

References

1.        Hohenfeld, I. Purification of histidine tagged bacteriorhodopsin, pharaonis halorhodopsin and pharaonis sensory rhodopsin 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.

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).