Light brings prosperity to the world.

Bioluminescence, as a fascinating phenomenon of living organisms, has attracted great interest among scientists. In recent years, researchers have successfully deciphered the mechanisms behind bioluminescence in various organisms. By employing synthetic biology techniques, they have successfully introduced genetic components related to the bioluminescent pathway of fungi into plant systems, endowing plants with the ability to emit light spontaneously . Applying green and low-carbon plant bioluminescence technology in daily life not only reduces energy consumption but also mitigates climate change, achieving carbon neutrality, which is a shared aspiration of humanity. Through the exploration of historical iGEM projects, multiple projects related to bioluminescence have been found. For instance, last year's "life bulb" project aimed to successfully incorporate fungal bioluminescent elements into algae.

After the transient transformation of the luminescent vector into the petals of Chrysanthemum, luminescence was observed 36 hours later. (Photos were taken by team secondary PI Lichuan Chen)

With more and more plants successfully undergoing luminescent modification, our team this year hopes to utilize luminescent plants for more useful purposes.

Through brainstorming and literature research, and after multiple rounds of discussions among team members, we hope to use luminescent plants as efficient and cost-effective biological sensors. As for the types of signals that our luminescent plant biosensors aim to detect, we initially had different opinions. Some team members wanted to use luminescence to detect oceanic nuclear radiation, while others wanted to monitor earthquakes. Some wanted to use luminescent plants to monitor nutrients such as N, P, K in soil, and provide timely reminders for farmers to fertilize and water their crops. There were also those who wanted to use luminescent plants as indicators of environmental pollution, which received agreement from the majority of team members. Each team member conducted extensive literature research based on their respective application ideas, in hopes of finding suitable genetic components for the modification of luminescent plants. In the end, we designed a promoter element (pZmPT7, BBa_K4799500) specifically responsive to phosphorus. From over 30 plant promoters that potentially respond to heavy metals, we further screened 12 gene promoters from Arabidopsis, corn, and tobacco, and designed primers for these promoters (hyperlink to part page). We plan to engineer these promoters into vectors containing appropriate expression elements, which were subsequently introduced into Escherichia coli and Agrobacterium through genetic engineering methods. Finally, we validated their expected functionality through transient plant transformation experiments.

Engineering considerations

In order to successfully obtain luminescent plants that can sense environmental signals, we need to address many knowledge gaps and engineering challenges. The biggest uncertainty we face is whether we can actually find genetic parts which are heavy metal-responsive.

Through literature research, we have found that genes specifically related to cadmium response have been reported in plants such as tobacco, tomato, Arabidopsis, and corn. Some genes have even had their cadmium response function validated through molecular experiments, including transcriptomic data and functional experimental data.

However, will the promoter that responds to heavy metals in specific plants show different behavior when transferred to model plants like tobacco is not sure?

To answer this question, we first used the commonly used GUS reporter system in plant functional gene research to verify the expression activity of the promoters we found in the literature that may respond to the heavy metal cadmium.

First DBTL Cycle

We tested more than 10 promoters, and all laboratory work for promoter pAtMRP3 was done by Youran Yao alone and listed below as an exemplar example of our engineering settings.



Our project's final goal is to engineer plants that respond to cadmium in the soil by glowing. To achieve this, we will first test whether the gene expression levels regulated by this promoter will change in the presence or absence of cadmium. Prior to testing this in a vector that contains the full set of the fungal luminescent vector, we will first test the AtMRP3 promoter in the traditional and classical GUS reporter gene device. Because the full set of the fungal luminescent vector contains 4 different genes, and the total size of the vector exceeds 20Kb, it is more difficult to insert our promoter into this system. More information on the fungal luminescence gene system can be found at the 2022 iGEM project "Life Bulb" by the team “ubc-okanagan” at https://2022.igem.wiki/ubc-okanagan. So, we used transient infiltration of Nicotiana benthamina leaves and GUS staining to test whether this promoter can achieve the expected function.

If feasible, we will then integrate it into the luminescent reporter gene. We chose the AtMRP3 promoter based on literature research, please see the listed reference below.


We will construct an Agrobacterium expression vector for pAtMRP3-GUS-NOS to test whether this promoter will respond to cadmium stimulation.
·First, we will search the gene information for AtMRP3 gene (AT3G13080) of Arabidopsis thaliana from the GenBank database and design primers to amplify the promoter region approximately 2 Kb upstream of the coding sequence. During the amplification of the promoter, we incorporate the homologous recombination arm sequences of the GUS vector, resulting in a promoter sequence with homologous arms.
·After performing homologous recombination, we will clone the construct into Escherichia coli, followed by single clone screening, bacterial liquid PCR, and sequencing verification.
·Once confirmed, we will extract the plasmid and transfer it into Agrobacterium competent cell (GV3101).
·Finally, we sprayed tobacco leaves with water and a solution containing 50 uM CdCl2, and then transiently infected the tobacco leaves with Agrobacterium carrying the pAtMRP3-GUS-NOS expression cassette. The response of the AtMRP3 promoter to Cd treatment will be evaluated by performing GUS staining.

All those sequencing result mapping are done by our supervisor Wanjun Chen.


The Phosphate Buffer Solution (PBS) was used as null control. The unedited super promoter (mannopine synthase promoter) was used as the positive control. AtMRP3-1 and AtMRP3-2 are two technical replicates. CdCl2 in the picture represents CdCl2 only sprayed on the tobacco leaves one day before transient infiltration. CdCl++ indicate both spraying CdCl2 solution to the leaves like the second column and watering CdCl2 solution into the soil three days before the infiltration.

In summary, pAtMRP3 will induce GUS gene expression with or without cadmium treatment, but if the tobacco were treated with a higher dose of cadmium, the expression was reduced compared to the pure water treatment.


Based on the results of this round of experiments, we have drawn the following conclusions and inspirations, which provide evidence for optimizing and improving subsequent experiments.

1.Based on the results, the GUS expression level decreased even further with cadmium treatment, and the discrimination ability of GUS was not high. Therefore, we plan to use an additional GFP expression vector reporter system to repeat the verification process.

2.Additionally, when the plants were sprayed with water, the promoter was highly expressed in the leaves. However, the expression level was reduced with cadmium treatment. To exclude the possibility that the high concentration of 50 μM cadmium used had damaged the plant cells, affecting the expression, we will consider increasing the concentration gradient in subsequent experiments. Furthermore, if Cd treatment really does reduce gene expression in the leaves, we can add a negative regulatory element or modify our luminescent plant reporting system, which normally emits light continuously but loses the ability of luminescence in the presence of heavy metal pollution.

3.Based on the experimental results for AtMRP3, we plan to continue searching for other available promoter sequences. This round of experiments builds up an effective testing system for finding heavy metal responding promoters.

4.To better understand the above results, We did more literature searches and found literature suggesting that the AtMRP3 gene can respond to cadmium only in the roots but is continuously expressed in the leaves. Therefore, it may be possible to use this promoter sequence in plants and use it to monitor heavy metal pollution in liquid culture systems or transparent gels.

Second DBTL Cycle


In our second round experiment design, we plan to initially attempt connecting our promoter pNtNRAMP2 separately with two different functional genes, GUS and GFP on two different carriers, to test whether our promoter can initiate the expression of these genes when the plant is exposed to a cadmium environment. We used transient infiltration of Nicotiana benthamina leaves to minimize the duration required for obtaining performance of pNtNRAMP2.

If feasible, we will then integrate it into the luminescent reporter gene. We chose pNtNRAMP2 based on literature research, please see the listed reference below.

Background of pNtNRAMP2

NRAMP (Natural Resistance-Associated Macrophage Protein) family members have been shown to be involved in the uptake and transport of metal ions in various plant species, from lower to higher plants. NRAMP proteins have 12 transmembrane domains and both N- and C- termini located intracellularly. NtNRAMP2 and NtNRAMP3 belong to the NRAMP gene family, and NtNRAMP3 has been experimentally proven to be responsible for the influx of Fe(2+), Mn(2+), Co(2+), Cu(2+), Ni(2+) and Cd(2+). Besides the response to metal ions,NtNRAMP3 also plays a general role in the regulation of the balance of several metals in plants grown under control conditions. As for NtNRAMP3 Promoter, it has two cis-regulatory elements which may involve in plant’s metal stress responses, MRE1 and IDE2([1]).

Similar functions have been found for NRAMP2 in other plants. The MsNRAMP2 gene of Alfalfa (Medicago sativa L.) responds to excess iron ions, and MsNRAMP2 responds in different ways in different plant parts. MsNRAMP2 Promoter has core elements, cis-acting elements of hormone response and multiple MYB- binding sites, which has an important role in the regulation of iron homeostasis([2]). As for potato, StNRAMP2 improves the accumulation of Cd(2+) in potato roots, stems, and leaves, but inhibits the accumulation of Cd in tuber([3]). Plus, TcNRAMP2, which belongs to Theobroma cacao, phylogenetically similar to NtNRAMP2 (identified as TheccNRAMP2 in the figure 1), is also involved in transport of Cd(2+.)([4]).

It has not been reported in literature whether gene NtNRAMP2 can respond to cadmium or not. We want to test it with our own engineering design.


We use the same building pipeline for the pAtPCR2 promoter in the first round of DBTL. That is, following promoter amplification, E.coli transformation, Agrobacterium transformation, and functional verification design. Particularly, based on the results of pAtMRP3, we add a GFP report experiment for NtNRAMP2 promoters to get more complete evidence on whether this promoter can be constructed using conventional methods , and respond to cadmium treatment.


pNtNRAMP2-GFP can be constructed successfully and pNtNRAMP2-GUS can respond to cadmium
pNtNRAMP2-GFP can be constructed successfully

Our promoter pNtNRAMP2 is designed to respond to cadmium stress. In order to locate the complete promoter sequence from the pNtNRAMP2 sequence, we amplified the first two thousand base pairs of the gene. We selected this specific range, as the promoter(s) of most genes would exist in the first two thousand base pair region.

PCR production of pNtNRAMP2-GFP is observed in the last lane on the right side, adjacent to the marker.
P2P is a Petri dish coated with NtRAMP2-GFP recombinant Escherichia coli.

After locating the promoter, we construct sticky ends for the promoter to pair up with new vectors. This is accomplished by adding two different pairs homology arms to the 5’ end of the promoter sequence. One pair comes from the vector GFP (green fluorescent protein), and the other one comes from the vector GUS (beta-glucuronidase). Finally , we successfully constructed pNtNRAMP2-GFP.

The electrophoresis results of homologous recombination of promoter pNtNRAMP2-GFP are marked at top of the figure below, we choose three tubes of those bacterial liquid with positive band for DNA sequencing, and the results show that the bacterial liquid sent for testing is homologous recombination.

pNtNRAMP2-GUS can respond to cadmium
groups pNtNRAMP2-GUS
clean water
CdCl2 (50ng/μL)
No-treatment Control
positive control
Conclusion pNtNRAMP2 can relatively significant response to cadmium

The results of our second round of experiment design helped us to find a brand-new cadmium-responsive promoter, which has never been reported to respond to cadmium in tobacco. We successfully constructed its GFP vector, and the sequencing verified that the connection was correct. At the same time, the response characteristics of its GUS vector to cadmium were tested, which is a very important discovery for us.


Our two round test results show that our vector construction method is feasible and widely applicable, and our functional verification experimental design can successfully help us to characterize the cadmium response characteristics of unknown functional genes. We will use this experimental method to carry out more and deeper experimental exploration, and will also share it with other teams in need in the same field.