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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）
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.
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.
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.
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.
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.
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.
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.
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.
| 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.