Our goal is to create auto-luminescent plants. To these ends, we need to find the luciferase genes that code bio-luminescent proteins and then find a way to transfer them into plants so the plant can glow and the lighter the better. Since our project involves gene transfers across different species, consideration of the different methods, plant species, and sources of genes we could potentially use was crucial to our experimental design.
1. Luciferase gene: Lux operon
The first step in creating glowing plants was to find a gene that codes for bioluminescence. After conducting literature research, we discovered several bacteria and animals that have bioluminescent systems: Aliivibrio fischeri, Alteromonas, Photohabdus, photobacteria, and fireflies. While all result in glowing, we chose Aliivibrio fischeri's lux operon, as the species is considered a key research organism when it comes to studies on bioluminescence and because its genes are easily obtainable.The lux operon is already in the registry.
2. A new part: LuxG
The lux operon found in Aliivibrio fischeri consists of the genes luxCDABEG. Each gene codes for a specific protein that contributes to the luciferase reaction. In an attempt to increase the intensity of light produced, we chose to insert another luxG gene into the plasmid, as the luxG gene provides reduced flavin mononucleotide, which is involved in the luciferase reaction.
3. Target plant: Nicotiana tabacum
Next, we had to decide which plants we would make glow. The options included tobacco plants, Arabidopsis, roses, Christmas trees, and even seaweed. While roses and Christmas trees were a popular choice since they are aesthetically pleasing and could be used as decorations, and while seaweed could create a pleasant effect if it could illuminate water, we decided on tobacco plants as they are readily available and are easier to conduct experiments on.
4. Gene Transfer Method: Agrobacterium-mediated transformation
After settling the genes we wished to transfer and the target plants, we had to decide upon how we would transfer the genes into plants. Gene transfer in plants is complicated by the presence of a cell wall. Literature research revealed several methods: agrobacterium-mediated transformation, biolistic gene gun, nanoparticle delivery, and so on. Though the gene gun was our first choice in the beginning, our research demonstrated that success rates are modest and that forceful bombardment could cause damage to plant tissues or lead to random integration of the DNA. Nanoparticle delivery, while capable of carrying a variety of payloads, is not well developed in plants. Ultimately, we chose an agrobacterium-mediated transformation as the agrobacteria are readily accessible and can deliver genes to areas of injection more feasibly than its alternatives.
5. Plasmid for agrobacterium-mediated transformation-pHB
As our final goal is to inject our plasmid with the lux operon genes into plants, we considered using vectors that have a promoter capable of initiating transcription in plants. pCAMBIA2301 and pCAMBIA1301 were two of the vectors we investigated as both of them possess a CaMV 35s promoter that has a high rate of transcription in transgenic plants. However, the direction of their promoters were awkward since the multiple cloning site with the restriction enzyme sites we wanted to use (EcoR1 and PstI/ HindIII) comes before the promoter, thus forcing the RNA polymerase to transcribe the whole plasmid (~11kb) before reaching the lux genes, which will drastically decrease transcription rates.
Therefore, we decided to use the pHB plasmid, which has the features shown in the picture below. The pHB plasmid has a 2x CaMV 35s promoter, which is capable of generating high levels of mRNA to cause greatly increased gene expression in plants. In addition, the MCS site comes directly after the promoter, and has the restriction enzyme sites that we can use (BamHI, PstI and HindIII).
1. Verify the BBa_K325909 part and confirm it works
According to the work of a previous team iGEM 10_Cambridge, BBa_K325909 could emit light in normal E. coli strains without the addition of any external substrate. We first need to transform its plasmid from the DNA Kit Plate (provided by iGEM) and to repeat the experiment in order to verify it. We need to make sure that the lux operon of this plasmid works.
2. Establish a method to compare bioluminescence strength in the bacteria
Measuring the bioluminescence strength at different time with different concentration of Arabinose can be used to establish a reliable method and induction condition to measure the bioluminescence strength.
3. Study the function of luxG and determine if it could improve the bioluminescence strength
Construct a new basic part, luxG, and a composite part with an extra luxG on the lux operon (luxCDABEG-luxG) to see if it could increase the bioluminescence strength as previous research has suggested. Measure the bioluminescence strength with an established method from step 2.
4. Construct pHB-luxCDABEG ang pHB-luxCDABEG-luxG plasmids
Insert luxCDABEG (lux operon) and luxCDABEG-luxG into pHB vectors, respectively.
5. Transformation target plasmids to agrobacteria
Transforming plasmids obtained from step 4 into agrobacterium and get agrobacterium containing the target plasmids.
6. Agrobacterium-mediated Transformation
Injection into Nicotiana tabacum (refer to experiment page for detailed protocol)
Wait for plants to glow and observe them on a daily basis 3-5 days after the injection of our plasmids.
Adem, Muhamed. “Recent Achievements Obtained by Chloroplast Transformation.” Plant Methods, Vol 13. BioMed Central. 2017.
Birky Jr., C. William. “The Inheritance of Genes in Mitochondria and Chloroplasts: Laws, Mechanisms, and Models.” Annu. Rev. Genet, Vol. 35, 125-148. University of Wisconsin-Madison. 2001.
Gaglani, Shiv. “Chloroplast Genetic Engineering: A novel method to produce therapeutic proteins.” Harvard Science Review. 2006.
GKrichevsky, Alexander et al. “Autoluminescent Plants.” PLoS ONE 5(11):e15461. 2010.
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.
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.
Thouand, Gerald and Robert Marks. “Bioluminescence: Fundamentals and Applications in Biotechnology – Volume 1.” Advances in Biochemical Engineering/Biotechnology. Springer. 2014.
Verma, Dheeraj. “Chloroplast Vector Systems for Biotechnology Applications.” Plant Physiology, Vol. 145, 1129-1143. American Society of Plant Biologists. 23 Nov 2014.