Using tobacco plants as our model organism, we aim to create autoluminescent plants capable of autonomous light emission without the addition of external substrates. To achieve this, we worked with the lux operon from the bioluminescent bacteria Aliivibrio fischeri and used it as our endogenous bacterial light-producing system, with additional characterization by the insertion of an extra luxG gene, which codes for the flavin reductase needed for flavin mononucleotide turnover. Working on the previous parts created by the 2010 Cambridge iGEM team, who had isolated the lux operon and placed it under an arabinose-induced promoter, we have conducted the transformation of part BBa_K325909, restriction enzyme digestion to cut out the lux operon from the plasmid, electrophoresis to separate DNA sequences of different sizes and obtain the part we want, gel extraction of the lux operon and ligation to connect the lux operon with the plasmid pHB, which contains a 2x 35s CaMV promoter capable of high transcription rates in plants, and finally Agrobacterium-mediated transformation to transfect tobacco plants with the plasmid pHB-luxCDABEG-luxG. In addition, we also conducted numerous gradient tests with different concentrations of arabinose in order to determine under which specific circumstances will the arabinose-induced luxCDABEG operon produce light with the greatest intensity, as additional characterizations of part BBa_K325909.
As China has one of the densest populations in the world, its consumption of electrical energy is the largest worldwide, with a yearly consumption of 5683 TWh. The energy used in lighting is one of the major contributors to the massive energy consumption. Therefore, we wanted to find a way to alleviate the energy usage using synthetic biology, which led us to the idea of autoluminescent plants. Even though we are now only at its early stages, when developed to its full potential, autoluminescent plants may be able to replace light bulbs, thus decreasing the demand for electricity not only in China but also throughout the world. Not only do autoluminescent plants conserve energy, but also create a more environmentally friendly world with less pollution and waste products. Although our project is not going to achieve energy conservation in an instant, it is the first step towards this future goal.
On the other hand, autoluminescent plants as decorations is a smaller but achievable goal according to current technology. As they do not require the input of additional substrates such as D-luciferin, as some of the previous light-emitting plants did, autoluminescent plants can be manufactured and sold as commercial products. In addition, through our research on websites such as Taobao.com, we have determined that there is a viable market for light-emitting plants. According to our research, luminescent wall stickers have monthly sales of more than 2500, and more than 1500 flower bouquets or vases are sold each month. This indicates that the autoluminescent plants we manufactured would have a market with a subset of consumers who are interesting in this specific field.
While previous research over the past decades have been done on bioluminescent plants, they utilized the firefly luciferase gene and require the addition of external substrates such as luciferin in order to produce light (Ow 1986; Miller 1992). However, transgenic plants possessing the bacterial bioluminescent light-emitting chemical pathway encoded for by the lux operon have also been shown to be able to produce a weak light using endogenous metabolic products and do not require the addition of substrates (Krichevsky 2010). Standard biolistic methods and homologous recombination sequences were utilized to insert the luxCDABEG genes along with the spectinomycin resistance selection marker aadA into the chloroplast genome of Nicotiana tabacum between the TrnI and TrnA genes (Krichevsky 2010). These transplastomic tissue were then cultured into an autoluminescent plant, which were capable of producing light visible by the human eye (Krichevsky 2010).
On the basis of previous research, our project is to create an autoluminescent plant using Agrobacterium-mediated transformation of our plasmid pHB-luxCDABEG and pHB-luxCDABEG-luxG in order to compare the function and effectiveness of the luxG gene, which codes for flavin reductase, in the production of light.
Krichevsky, Alexander et al. “Autoluminescent Plants.” PLoS ONE 5(11):e15461. 2010.
Ow, David et al. “Transient and Stable Expression of the Firefly Luciferase Gene in Plant Cells and Transgenic Plants.” Science, Vol. 234, 856-859. 14 Nov 1986.
Millar, Andrew et al. “A Novel Circadian Phenotype Based on Firefly Luciferase Expression in Transgenic Plants.” The Plant Cell, Vol. 4, 1075-1087. American Society of Plant Physiologists. Sept 1992.