Today's climate crisis has become an urgent issue of global concern, causing profound impacts on human health, ecosystems, economies, and societies.
Global temperatures are continuously rising, extreme weather events are occurring frequently, sea levels are on the rise, acid rain pollution is a concern, the ozone layer is depleting, and biodiversity is declining sharply.
All of these issues stem from human activities and the resulting emissions of greenhouse gases. In China alone, during the first half of 2021, coal consumption for electricity generation reached a staggering 1.57 billion tons, and natural gas consumption for electricity generation amounted to 182.7 billion cubic meters, according to data from the National Energy Administration. Lighting electricity consumption accounts for 10% to 12% of China's total electricity generation, and the production complexity and recycling challenges of LED lights and clean energy lighting fixtures are issues that need attention.
In the East China Sea region of China, there exists a bioluminescent organism known as Noctiluca scintillans, which emits a blue glow upon contact and is affectionately called "blue tears." We imagine that if this carbon-negative algae that can convert solar energy can be used for lighting, it will definitely help us solve the climate crisis and energy crisis.
However, the problem is that the algae themselves are the product of eutrophication pollution of the water body, and ordinary culture conditions cannot allow them to become a reliable source of continuous light.Synthetic biology gives us new possibilities. We can integrate luciferin systems on carbon-fixing algae chassis and synthesize our own noctiluca. This is how the LAMPS project was born.
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Our original idea was to transfer the luxCDABE operon gene into PCC7942 strain to achieve our basic goal of making algae glow. The luxCDABE operon encodes the fatty aldehyde transferase luxD, fatty acid reductase luxCE, and fatty aldehyde oxidase luxAB. Because luxAB emits blue light (490 nm) during oxidation, it was once widely used in microbial experiments as a reporter.
We expected, as the results of the experiment, that the light emitted by the primitive lux operon was a very faint blue light (similar to a schematic diagram of PV), which is very technological, but far from being called "LAMPS". Our project first set out to make our enzymes emit brighter and more colorful light for practical applications. We use both protein engineering and metabolic engineering to achieve the design goal of increasing light intensity.
Since the LuxCDABE system itself has a low brightness and is not enough to be used in real life, we decided to increase its brightness by some other methods. After reviewing the literature, we found that when a fluorescent protein cp157Venus was fused to the C-terminal of the LuxB protein, the brightness of the Lux operon system was significantly increased and the color of the light changed due to the Biofluorescence Resonance Energy Transfer (BRET) between LuxA, LuxB and Venus. So we constructed a plasmid containing the LuxB:Venus gene and expressed it. [1]
However, since the brightness enhancement still fell short of our requirements and we wanted a richer color system, we screened and expressed brighter fluorescent proteins from FPbase (www.fpbase.org) that met the requirement of excitation wavelength.
However, since many fluorescent proteins only have sequence data but not fluorescence data, in order to identify whether their fluorescence performance is up to standard or not, we used recurrent neural networks to establish a mapping model from sequence to function, which helped us to select fluorescent proteins with incomplete information.
In the subsequent experiments, it was also proved that the model predicted more accurately. In addition, in order to break through the shackles of nature to create stronger fluorescent proteins, we used the Generative Adversarial Network model to generate sequences of fluorescent proteins that are not found in nature, which are predicted to have stronger brightness after simulation.
The fluorescence reaction substrate is normally increased by the introduction of LuxF and luxG proteins.
Additionally, the intensity of light is an essential criterium of LAMPS, leading us to involve two other parts: LuxF and LuxG. Previous study has shown that LuxF would activate luciferases (consists of LuxA and LuxB) by binding to the inhibitor of luciferases [2]. And LuxG also contributes to the luminescence with extra substrates FMNH2 [3]. In a conclusion, in this section we combined basic lux operon, luxG and luxF to form luxCDABEGF and proved the composite part enabled E.coli to emit stronger luminescence than the ordinary lux operon, which is the basis of our whole design LAMPS.
In addition to better realizing the fluorescence function itself, we further explored the design of synthetic biology and biocybernetics in enhancing the application capabilities of engineering cyanobacteria.
We hope to make LAMPS "smart", capable of automatically turning on after entering the night and automatically turning off during the day without the need for manual control. Fortunately, cyanobacteria, as photoautotrophic organisms, have an inherent biological clock rhythm system. Through a series of protein interactions and phosphorylation modifications, it can generate oscillatory outputs in downstream promoter PkaiBC with a 24-hour cycle. [4,5]
Connecting the lux luminescent gene cluster downstream of PKaiBC allows for diurnal and nocturnal rhythmic expression of the fluorescent genes.
As obligate photoautotrophic organisms, cyanobacteria's energy metabolism is largely self-sustaining, and it is particularly challenging to maintain the expression of a large amount of exogenous proteins at night. Therefore, adjusting metabolism to meet the fluorescence emission needs of LAMPS is important to considerate.
Metabolic optimization involves several aspects:
1. We expressed sRNA targeting Glgc, which is a gene encoding glucose-6-phosphate translocase. It reduces the expression of glucose-6-phosphate translocase, thereby decreasing the process of glucose-6-phosphate conversion into ADP-glucose and glycogen synthesis, allowing more material to flow into sucrose synthesis and increasing photosynthetic efficiency. [6]
2. Additionally, we learned from the article [7] that cyanobacteria have a much higher concentration of pyruvate than acetyl-CoA, suggesting significant untapped metabolic potential. We plan to express pyruvate dehydrogenase to break down more pyruvate into acetyl-CoA, releasing this untapped metabolic potential and increasing product expression [7].
Furthermore, considering biosafety, we designed a killing switch to prevent leaks. This is also a fully automated system composed of a logic circuit centered around a metal ion-responsive promoter.
We expressed nuclease A(nucA) under a constitutive promoter and linked its inhibitor gene, nuiA, downstream of a Ni ion-responsive promoter. When a certain concentration of Ni ions is added to the culture medium, it induces continuous expression of nuiA, allowing the microorganisms to survive. However, once the microorganisms leak and leave the culture medium, the concentration of Ni ions in the environment decreases, nucA is derepressed, and it cuts the DNA of cyanobacteria, leading to their death. [8] This design achieves automatic sterilization in the event of a leak and eliminates the need for manual handling.
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. The wedding needs to decorate the outdoor lawn, you can use our natural degradation does not need to recycle the small luminous ball, a sprinkle on the ground, you can continue to glow for a few days, and do not need to recycle, the grass will be restored in a month (even more lush grass).
In the water park at night, people wear luminous bracelets, throw luminous volleyballs on the water, children's swimming rings are luminous, and luminous ducklings float on the water. This is also a scenario that can be realized with our technology.
The light-emitting ball could also be used as a marker for emergency relief. When firefighters enter the forest to look for missing tourists, they can drop the ball along the route up the mountain, so they can quickly mark the route.
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.
These two applications amplify the product benefits of biomaterials and combine the technical characteristics of LAMPS. This product makes our technology commercially viable and provides a promising value conversion path for this technology.
[1] Kaku T, Sugiura K, Entani T, Osabe K, Nagai T. Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer. Sci Rep. 2021;11(1):14994. Published 2021 Jul 22. doi:10.1038/s41598-021-94551-4
[2] Brodl, Eveline et al. “The impact of LuxF on light intensity in bacterial bioluminescence.” Journal of photochemistry and photobiology. B, Biology vol. 207 (2020): 111881. doi:10.1016/j.jphotobiol.2020.111881
[3] Nijvipakul, Sarayut et al. “LuxG is a functioning flavin reductase for bacterial luminescence.” Journal of bacteriology vol. 190,5 (2008): 1531-8. doi:10.1128/JB.01660-07
[4] 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.
[5] 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.
[6] 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.
[7] 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.
[8] 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.
