In order to make the cyanobacteria chassis and luciferase system meet the requirements of practical applications, we have conducted more design work. Including adjusting the metabolism of cyanobacteria, using BRET to improve luminescence, expression control based on rhythmic promoters, and suicide switches, etc. LAMPS under the full-circuit design can exert its functions in their respective places and has greater application potential, so we have conducted auxiliary design for more application scenarios.
We use the chassis Synechococcus elongatus, a type of cyanobacterium widely employed as a model organism for metabolic research and genetic modification. However, this strain is incapable of maintaining exogenous plasmids. We employ homologous recombination by connecting two homologous arms at both the target gene's and the marker gene's ends to integrate the target genes into the cyanobacterium's genome.
Currently, three neutral insertion sites have been identified as NSI, NSII, and NSIII, altering where does not affect the cyanobacterium's own metabolism, making it an ideal insertion site.
PCC7942 is an improved strain of S. elongatus that can autonomously uptake exogenous DNA sequences. Transformation can be achieved by incubating the bacterial culture mixed with the plasmid overnight.
One of the major applications of LAMPS technology will be as an environmental facility that directly serves human life. Whether it is luminous markers, garden light sources, or ambient lighting, it will be very convenient to turn off during the day and open at night, directly solving the trouble of users who need to interact or add chemical reagents, etc., and at the same time make our products more durable, easy to use and sustainable.
Our first design on the gene circuit shows why the chassis of LAMPS must not be Synechococcus elongatus PCC7942. In addition to being a biological version of the photochemical transducer platform, the innate rhythm system has brought great help to our gene circuit design. Using pKaiBC and pPsbA promoters, this delicate biomolecular oscillation system can be held hostage to output protein expression with circadian rhythms.
We hope to make LAMPS "smarter", capable of automatically turning on after entering the night and automatically turning off during the day without the need for manual control. We need a gene expression system that responds differently to day and night. Fortunately, cyanobacteria, as photosynthetic autotrophs, have an inherent circadian system. This system consists of several Kai proteins (pronounced "kai," which means "cycle" in Japanese) and a series of phosphorylation and interactions with auxiliary factors.[1]
During the daytime, the KaiC protein undergoes continuous phosphorylation and interacts with SasA, which enhances the promotion of the downstream promoter PkaiBC, reaching its peak in the evening. At night, KaiC protein gradually dephosphorylates, SasA dissociates, and it exerts an inhibitory effect on the PkaiBC promoter. The periodic expression of PkaiBC, in turn, influences KaiC, forming a stable cycle that can be maintained for up to a week even in vitro[2].
PkaiBC is the direct downstream output of the cyanobacterial circadian clock. We can connect the genes responsible for fluorescence expression with the PkaiBC promoter to achieve periodic regulation of luminescence.
Biological systems subject to this regulation are able to express serial proteins during the day to store substrates needed for fluorescence oxidation, while expressing luxAB fusion proteins at night.
During the day, the pPsbA promoter expresses luxCDEFG to store energy substances, reducing substrates, and other "fuels". luxD converts Myristol-ACP into fatty acids, and LuxCE reduces these fatty acids to fatty aldehydes. LuxF would activate luciferases (consists of LuxA and LuxB) by binding to the inhibitor of luciferases . And LuxG also contributes to the luminescence with extra substrates FMNH2 .
At night, pKaiBC initiates the expression of luxAB fusion protein. Fluorescent proteins fused after the native version of the luxB subunit naturally form the BRET system with luxAB. The brighter multicolored biofluorescence lights up promptly in the evening.To make the bacterial LuxCDABE fluorescence system brighter and more useful for practical applications, we utilized biofluorescence resonance energy transfer (BRET) to increase its brightness and alter its color. [1]We created a new fusion fluorescent protein called LuxB:cp157Venus by attaching the yellow fluorescent protein cp157Venus to the C-terminal of LuxB using a linker (Glu-Leu). Since the emission spectrum of the LuxA-LuxB complex overlaps with that of the excitation spectrum of cp157Venus to a certain extent, when the two are close enough to each other (≤10 nm), the LuxA-LuxB complex in the excited state is able to undergo dipole-dipole resonance with cp157Venus, transferring its own energy to the latter in a non-radiative manner, causing the latter to emit light with different frequencies and amplitudes. Since the efficiency of BRET is related to the sixth power of the distance between the two only when the distance between the fluorescence donor and the fluorescence acceptor is appropriate, it is able to change the wavelength of the light while significantly increasing the brightness.
As strict photoautotrophic organisms, cyanobacteria have a basic self-sustaining energy metabolism[1], and it is difficult to maintain a large amount of exogenous protein expression at night. Therefore, we plan to optimize the metabolism of cyanobacteria, allowing them to accumulate sufficient substances and energy through photosynthesis during the day to supply nighttime illumination.
To increase the accumulation of luciferin substrates, we have split the luxCDABEFG gene cluster and connected it to different promoters. The luxA and luxB genes, responsible for expressing the luciferase enzyme luxAB dimer, are connected downstream of PKaiBC, achieving periodic expression of the luciferase enzyme. And genes involved in the production and recycling of luciferin substrates, such as luxC, luxD, luxE, luxF, and luxG, are all linked downstream of the constitutive strong promoter PpsbA. This promoter is highly expressed during the daytime, resulting in the significant accumulation of substrates in cyanobacteria.
To enhance energy reserves and increase the carbon flux in cyanobacteria for the expression of luciferase genes, we have employed the Hfq companion protein and siRNA to silence the expression of the glgc gene, which encodes D-glucose-6-phosphate translocase. This blocks the conversion of glucose-6-phosphate into ADP-glucose, preventing further synthesis into glycogen, thereby reducing glycogen content and increasing sucrose production.[2]
Furthermore, through article research, we have learned that cyanobacteria have a very high content of pyruvate, approximately 100 times that of acetyl-CoA. This untapped energy source holds significant metabolic potential. We have designed the use of constitutive promoters in cyanobacteria to express pyruvate dehydrogenase. This will increase the conversion of pyruvate into acetyl-CoA, ultimately enhancing bio-production.[3]
Our product is typically stored in sealed containers, but we need to consider the potential for leakage in case of container breakage. Therefore, we have designed a killing switch that mainly relies on nucA endonuclease[1]. This is also a fully automated system consisting of a logic circuit based on a metal-ion-responsive promoter as its core. Downstream of a constitutive promoter, nucA is connected, and downstream of the metal-ion-responsive promoter, an inhibitor of nucA called nuiA is connected. Considering cyanobacteria's tolerance to Ni ions, we use PnrsB-BCD, a nickel-responsive promoter, to initiate the expression of nuiA. When a certain concentration of Ni ions is added to the culture system, nuiA is expressed in the solution environment, thereby inhibiting the activity of nucA. However, once cyanobacteria are removed from the culture system, the concentration of Ni ions in the environment decreases, leading to a decrease in nuiA expression. This, in turn, removes the inhibition of nucA, resulting in the cleavage of cyanobacteria's genomic DNA, achieving self-destruction.
Considering the commercial feasibility and the value transformation of the technology, we worked with industrial design students to do some scenario exploration and product development with our technology.
Reference: images from here
This product is suitable for atmospheric lighting in weddings/events/private villas.
Or entertainment toys in water parks at night.
The light-emitting ball could also be used as a marker for emergency relief.
In addition to the biodegradable balls, LAMPS can also produce products that can be used in a wide range of Settings: the roofs of public buildings such as stations/pavilions/corridors/flyovers, floor signs in parks/green Spaces/exhibitions, lighting in historic wooden buildings or waterfront areas, installation art, costume design or experimental art.
Considering the instability of algae organisms and the harsh requirements of the environment, we also designed a portable and easy-to-use detection & maintenance equipment that can detect ambient temperature and humidity, luminescence intensity of algae and bacterial density, and reflect the data on the LED screen in real time.
[1] Snijder J, Axmann IM. The Kai-Protein Clock-Keeping Track of Cyanobacteria's Daily Life. Subcell Biochem. 2019;93:359-391. doi: 10.1007/978-3-030-28151-9_12. PMID: 31939158.
[2] Chavan AG, Swan JA, Heisler J, Sancar C, Ernst DC, Fang M, Palacios JG, Spangler RK, Bagshaw CR, Tripathi S, Crosby P, Golden SS, Partch CL, LiWang A. Reconstitution of an intact clock reveals mechanisms of circadian timekeeping. Science. 2021 Oct 8;374(6564):eabd4453. doi: 10.1126/science.abd4453. Epub 2021 Oct 8. PMID: 34618577; PMCID: PMC8686788.
[3] Tan LR, Cao YQ, Li JW, Xia PF, Wang SG. Transcriptomics and metabolomics of engineered Synechococcus elongatus during photomixotrophic growth. Microb Cell Fact. 2022 Mar 5;21(1):31. doi: 10.1186/s12934-022-01760-1. PMID: 35248031; PMCID: PMC8897908.
[4] Li, S., Sun, T., Chen, L., and Zhang, W. (2021). Designing and Constructing Artificial Small RNAs for Gene Regulation and Carbon Flux Redirection in Photosynthetic Cyanobacteria. Methods Mol Biol 2290, 229-252.
[5] Hirokawa Y, Kubo T, Soma Y, Saruta F, Hanai T. Enhancement of acetyl-CoA flux for photosynthetic chemical production by pyruvate dehydrogenase complex overexpression in Synechococcus elongatus PCC 7942. Metab Eng. 2020 Jan;57:23-30. doi: 10.1016/j.ymben.2019.07.012. Epub 2019 Aug 1. PMID: 31377410.
[6] Čelešnik H, Tanšek A, Tahirović A, Vižintin A, Mustar J, Vidmar V, Dolinar M. Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium Synechocystis sp. PCC 6803. Biol Open. 2016 Apr 15;5(4):519-28. doi: 10.1242/bio.017129. PMID: 27029902; PMCID: PMC4890671.
