iGEM 2024 — Team nmu-china

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each
day for your project.{% endblock %}
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### Notebook

Scroll To Explore

# 1.Model

# 2.Human Practices

# 3.Experiments

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one
iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{%
endblock %}
{% block page\_content %}

### Engineering Success

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# Overview

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-1.png)

In the article " Can a biologist fix a radio? ", a reflection on the research methodology of biology is
presented, hinting at the close integration of biology and engineering in the field of synthetic biology.
The Engineering Cycle is the core framework for the development of synthetic biology projects, with
aniterative approach to optimising designs, validating hypotheses and improving the overall performance of
the project. Adhering to the above concepts, our team integrates engineering thinking into different
functional modules of the project, and sets up the Engineering Cycle for continuous iteration to ensure the
compatibility and operability between the modules. We strictly follow the phases of design, build, test, and
learn, and continue to improve the project solution in multiple aspects. In the design phase we clarified
the core objectives of the project and proposed innovative design ideas by combining literature research and
expert advice. In the construction stage, we sent the designed gene fragments to the company for synthesis,
and the synthesised plasmids were transferred to the chassis cell. We paid attention to the accuracy and
reproducibility of the construction, and made a detailed experimental procedure to ensure the consistency of
the experiment. Thus, in the testing phase, we rigorously verified whether the construction of the module
was feasible through experiments. And in the learning phase, we analysed the test results and identified the
deficiencies and room for improvement in the design. After determining whether the project should continue
to be refined or not, the ideas were integrated into the next cycle. We objectively viewed successes and
failures in each engineering cycle, optimised our project to the best of our ability, and constructed safe
and consistently effective light-emitting engineering bacteria.

Our light emitting module includes 5 cycles, in cycle1 and 2 we successfully verified the feasibility of the luminescence genetic circuits. Cycle3 focused on making the engineered bacteria luminescence after entering seawater. Cycle4 solved the problem of luminescence substrate and energy waste, and the engineered bacteria luminescence as much as possible in the night, and cycle5 is the strategy we proposed to further increase the luminescence intensity.

The safety module also includes 5 cycles, in cycle1 we choose the right bacteria for the chassis. Cycle2 solved the problem of normal survival and functioning of engineered bacteria in the ocean and space environment. Cycle3 provided a good strategy for the preservation of the product. Cycle4 proposed a solution to the problem of false positives and contamination that may be caused by the leakage of engineered bacteria. Cycle5 focused on the strategy of recycling and disposal of the product after use.

* [**Light Emitting Module**](#Lc1)
  + [Cycle1: Fundamental luminescence](#Lc1)
  + [Cycle2: Enhanced luminescence](#Lc2)
  + [Cycle3: Osmotic receptivity](#Lc3)
  + [Cycle4: Night selectivity](#Lc4)
  + [Cycle5: Aggregative luminescence exploration](#Lc5)
* [**Safety Module**](#Sc1)
  + [Cycle1: Strain selection](#Sc1)
  + [Cycle2: Microbial survival](#Sc2)
  + [Cycle3: Product preservation](#Sc3)
  + [Cycle4: Preventing false positives and leakage](#Sc4)
  + [Cycle5: Product recovery](#Sc5)
* [**References**](#references1)
* ![糖豆人](https://static.igem.wiki/teams/5210/logo-astronaut-2.png)

  ![](https://static.igem.wiki/teams/5210/logo/logo-5.png)

# Cycle1: Fundamental luminescence

## 1.Research:

We summarized the luminescence mechanism of several luminescent bacteria and found
that almost all luminescent bacteria rely on the Lux gene cluster for
bioluminescence. LuxA and B encode the α and β subunits of luciferase, respectively,
and luxC, luxD, and luxE encode NADPH-dependent LuxC, luxD, and luxE encode
NADPH-dependent fatty acid reductase (54 kDa), acyltransferase (33 kDa), and ATP
synthase (42 kDa), which together form the fatty acid reductase complex that
produces long-chain aliphatic aldehydes as the electron donor for luminescence
reactions. While luxI and luxR upstream of luxC are bacterial Quorum sensing,
regulating the expression of downstream luxCDABE genes. In the natural environment,
the population density of luminescent bacteria needs to reach a certain threshold to
produce fluorescence.

## 2.Imagine:

We plan to engineer bacterial bioluminescence using Lux gene clusters. However, the
population density requirement caused by Quorum sensing regulation is not beneficial
to our project: ①Bacteria need to grow to a certain population density to produce
fluorescence, which slows down the rate of luminescence. ②It is difficult to
accumulate auto-inducers (AIs) in the ocean. Therefore, we decided to introduce only
luxCDABE luminescent gene clusters, discarding the upstream Quorum sensing
regulation part.

## 3.Design and Build:

We constructed a prokaryotic expression vector containing LuxCDABE and validated it
in the lab using BL21 (DE3) in the hope of achieving bioluminescence in making
engineered bacteria.

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-2.png)

## 4.Test:

we cloned the synthesised luxCDABE fragment onto the pet-28a plasmid. Subsequently,
we transformed the plasmid and blank pet-28a plasmid into E. coli BL21,
respectively. After seeding cells and overnight incubation, we extracted equal
volumes of bacterial cultures using 0.5 mM IPTG induced at 12°C for 10 h.
Subsequently, luminescence was performed for naked eye observation and luminescence
test was performed using a microplate reader.

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-3.png)

## 5.Learn:

Unfortunately, we did not observe luminescence with our naked eye in the preliminary
experiments. After adjusting the IPTG induction concentration and time we reran the
experiment, but the results were still unsatisfactory. Analysing the results of the
experiment we believe that the gene line was the cause of the failure, and in the
next cycle we will improve the existing gene line.

# Cycle2: Enhanced luminescence

## 1.Research:

In order to optimise the gene line, we conducted a lot of literature research and
browsed past iGEM teams, and finally found that the 2023 Shanghaitech\_China team
also used the lux gene cluster and improved it by introducing luxF and G to enhance
the luminescence intensity. In addition, for luxF and G, we found literature
supporting that they can enhance the luminescence intensity of luxCDABE
[1,2].

## 2.Imagine:

We plan to introduce luxF, G to enhance the luminous intensity of engineered
bacteria.

## 3.Design and Build:

Before we planned to design the plasmid, we contacted Shanghaitech\_China and got the
pet28a-LuxCDABEFG plasmid as a gift from Shanghaitech\_China, we are very grateful to
them for their support!

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-4.png)

## 4.Test:

We performed the same experiment as Cycle1 to test the luminescence ability of the
optimised line, and the results show that our optimisation was successful! We
successfully observed the blue fluorescence with the naked eye! After observing the
fluorescence with the naked eye, we performed a luminescence test with a 15s long
exposure shot and a versatile microplate reader.

![Gut-sweeper](https://static.igem.wiki/teams/5210/result-17.jpg)

## 5.Learn:

Through the optimisation of the gene line and the introduction of luxF and G, we
successfully made the engineered bacteria emit blue fluorescence and achieved the
expected goal. However, we are not satisfied with this, in order to further verify
the area and intensity of luminescence that our engineered bacteria can achieve in
real-life applications, the modelling group will carry out modelling simulations.

# Cycle3: Osmotic receptivity

## 1.Research:

Making the engineered bacteria glow at the right time is another key issue after
successfully making the engineered bacteria capable of luminescence, and either too
early or too late luminescence can cause negative impacts in the practical
application of the project. In order to achieve accurate tracing without affecting
the correct judgement, we need to modify the engineered bacteria so that they emit
light quickly when they sense a distressing environment.

## 2.Imagine:

The most notable feature of seawater is the high osmotic pressure, can we make the
engineered bacteria start to glow as soon as they hit the water and feel the high
osmotic pressure?

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-6.png)

## 3.Design and Build:

We conducted a literature review and found that bacteria can sense changes in
external osmotic pressure via the EnvZ/OmpR two-component system [3]. The
EnvZ/OmpR system is prevalent in Gram negative bacillus, and EnvZ regulates OmpR by
sensing changes in osmotic pressure, which subsequently affects the expression
levels of membrane pore proteins OmpC and OmpF expression levels. By constructing
gene circuits, we could achieve to make the engineered bacteria glow in a high
osmotic pressure environment i.e., they start to glow after falling into water.

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-7.png)

## 4.Test:

We verified the functionality of the two-component system in the laboratory using
BL21(DE3). We ligated the reporter gene downstream of Pompc and configured the
medium with different NaCl concentrations to culture the engineered bacteria. The
ability of the EnvZ/OmpR two-component system to sense osmotic pressure in the
external environment was verified by observing the reporter gene expression of the
engineered bacteria after incubation in medium with different NaCl concentrations.

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-8.png)

## 5.Learn:

Our experimental results show that increasing osmolarity effectively increases
reporter gene expression over a range of osmotic pressures, suggesting that our
genetic circuit design was successful in achieving the desired results. However, we
saw a decrease in reporter gene expression as osmolarity continued to increase. This
is because the BL21(DE3) we used in the lab is not tolerant to high osmolarity, and
its activity decreases in high osmolarity environments, resulting in a decrease in
gene expression. The chassis cell we chose for our project is Vibrio natriegens,
which can survive in high salt environment and is expected to achieve better
results.

![Gut-sweeper](https://static.igem.wiki/teams/5210/result-18.png)

# Cycle4: Night selectivity

## 1.Research:

Our PI pointed out that our engineered bacteria mainly play a tracer role at night,
and it seems redundant to emit light during the day, so we can consider
proliferation and substrate and energy accumulation during the day to enhance the
intensity of light emission at night. According to the expert's opinion, we analyse
the role of each gene in the lux gene cluster and look for optogenetic tools.

## 2.Imagine:

We hope to separate luxCDEFG, which generates fluorescent substrate, from luxAB,
which encodes luciferase, and introduce optogenetic tools so that luxAB is expressed
only in the dark environment, while luxCDEFG is not regulated by light, and
continues to accumulate substrate, to achieve the purpose of conserving substrate
and energy, and enhancing the intensity of luminescence.

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-10.png)

## 3.Design and Build:

We introduced the UV-induced promoter PumuDC and repressor protein CI, downstream
luxAB expression is repressed when UV intensity is high during the daytime, and when
UV decreases at night the repressor effect also decreases, and luxAB expression
starts to glow. While the rest of the genes are not regulated by light, which means
that substrate accumulation does not stop.

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-11.png)

## 4.Test:

We characterised the UV promoter PumuDC in the laboratory by assaying reporter gene
expression.

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-12.png)

However, the results of the experiment were not satisfactory, and prolonged UV
irradiation did not have the desired effect of increasing the expression of the
reporter gene.

## 5.Learn:

By reflecting on the process and results of the experiment, we found that too much UV
light was the main reason for the failure, and that too much UV light killed the
bacteria more than it initiated gene expression.

Before adjusting the UV intensity to continue the experiment, we reflected and
analysed the genetic circuit again. PumuDC is a classical promoter associated with
the DNA damage response in prokaryotes (especially E. coli), and it plays an
important role in the SOS response in bacteria. In the absence of DNA damage stress,
such as UV light, PumuDC is repressed by the LexA protein, which, as a key repressor
of the SOS response, binds to specific DNA sequences in the SOS-associated promoter
and represses the transcription of SOS-responsive genes. When bacterial DNA is
damaged by factors such as UV light, ssDNA accumulates. the RecA protein recognises
and binds to this single-stranded DNA, forming the RecA-ssDNA complex. This complex
activates the protease function of RecA and promotes the self-cleavage of the LexA
protein. with the self-cleavage and degradation of the LexA protein, the inhibition
of PumuDC is lifted.

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-13.png)

Our review of the literature revealed that for PumuDC activation, UV-C radiation is
the most effective, significantly inducing SOS responses in the range of 10-50 J/m²
[4]. However, in the natural environment, UV-C is almost entirely
absorbed by the atmosphere and cannot reach the ground. Strong sunlight can indeed
activate the PumuDC promoter via UV-B, but the activation effect is weaker compared
to that of direct UV-C light sources in the laboratory. The effect of sunlight
activation is limited by the UV-B content in the atmosphere and the exposure time,
and it usually takes a longer period of direct sunlight exposure to achieve a The
effect of sunlight activation is limited by the UV-B content in the atmosphere and
the exposure time.

Therefore, this promoter was not suitable for our project, and through further review
of the literature, we found an optogenetic tool for far-red light-regulated
transcriptional activation apparatus that has been well characterised
[5].

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-14.png)

# Cycle5: Aggregative luminescence exploration

## 1.Research:

In order to further enhance the luminescence intensity, we interviewed Prof
Junlong Zhao. Prof Zhao believes that the expansion of luminescence
intensity and area requires the use of marine native luminescent bacteria, rather
than relying solely on the proliferation of engineered bacteria to produce
luminescence. This is in line with the "in situ resource utilisation" concept of
iGEM's Space villiage, but here we are using in situ resources from the ocean rather
than from outer space.

## 2.Imagine:

We hope to cause Vibrio fischeri in the ocean to aggregate and fluoresce, further
increasing the intensity and area of luminescence.

## 3.Design and Build:

We investigated how Vibrio fischeri move in the ocean and found that Vibrio fischeri
are predominantly chemotactic. Vibrio fischeri sense changes in chemicals in their
surroundings and respond with chemotactic movements, which help them to find
nutrient-rich areas or avoid harmful substances. Chemotaxis relies on methyl
receptor chemotactic proteins (MCPs) to sense chemical gradients, which influence
bacterial motility by redirecting the rotation of the flagellum through a series of
signal transduction pathways.

We wanted to achieve the aim of aggregating Vibrio fischeri in the ocean by adding
chemotaxis to the lyophilised bacterial powder, the chemotaxis that were included
for consideration were:

* Amino acids (glutamic acid, alanine, etc.)
* Sugars (glucose, lactose, etc.)
* Fatty acids (oleic, stearic, etc.)
* N-Acyl homoserine lactone (AI-1)

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-15.png)

## 4.Test:

Analysing the chemotactic compounds we chose, most of them are nutrients except for
the Quorum sensing molecule N-acyl homoserine lactones (AHLs), which, while allowing
Vibrio fischeri to aggregate through chemotaxis, are also chemotactic for other
marine bacteria, including a number of Pathogenic bacteria (Vibrio parahaemolyticus,
Vibrio cholerae), causing these bacteria to aggregate is an undesirable outcome that
poses a greater risk.

## 5.Learn:

We discarded the use of nutrients as chemotaxis agents and continued to review the
literature with the aim of exploring chemotaxis agents that can specifically
aggregate Vibrio fischeri. We found that N-acetylneuraminic acid and (GlcNAc)2 in
chitosan, as substances that help Vibrio fischeri to colonise the squid, have a
specific and strong attraction to Vibrio fischeri, and we decided to use them as
chemotaxis agents [6,7].

In addition, we believe that this strategy requires a certain amount of Vibrio
fischeri in the oceans (especially at the surface), and in the future we will
further investigate this data in different parts of the planet and optimise our
project.

# Cycle 1: Strain selection

## 1.Research:

What are the conditions that our engineered bacteria have to fulfil in order to meet
search and rescue needs?

Our HP investigation of the fluorescent sea phenomenon found that it was due to the
proliferation of Dinoflagellates, represented by Noctiluca, to the point of strong
fluorescence, suggesting that nutrient factors such as phosphorus, potassium and
iron are elevated in the seawater in this area, allowing bacteria, cyanobacteria and
unicellular algae, which are used as food by the Dinoflagellates, to Dinoflagellates
as food for bacteria, cyanobacteria and unicellular algae. This is one of the
precursors of seawater eutrophication, which, if it develops further, may result in
an explosive growth of Dinoflagellates, leading to the formation of red tides.

We investigated and analysed the principle of luminescence, and found that the
luminescence of Noctiluca originates from its intracellular luciferin-luciferase
reaction. Luciferin is a class of small molecules, which can be used as a substrate
to bind to luciferase, and luciferase uses ATP to make the luciferin release
luminescence, completing the conversion of chemical energy to light energy. In
addition, a number of bacteria are also capable of similar bioluminescence through
the luciferin-luciferase reaction.

Since our engineered bacteria will be used in rescue missions, in addition to being
able to emit light we also need the engineered bacteria to be able to proliferate
and emit light quickly after the user is in distress. In addition, due to the
uncertainty of the status of the rescue target, the chassis cell we choose should be
non-pathogenic to human body. Combined with the above, our project firstly considers
the safety of the rescuers, not only to ensure that the engineered bacteria can emit
light to play the localisation effect, but also not to cause additional harm to the
rescuers. This requires us to find a chassis cell that can meet the above
conditions.

## 2.Imagine:

Can we use safe bioluminescent phenomena for tracing?

In our research phase, we have already discovered some autoluminescent organisms, and
the mechanism of luminescence of some of the organisms discovered in the preliminary
stage has been applied in the field of synthetic biology, we can start with
autoluminescent organisms and examine their advantages and disadvantages as project
chassis cells, and then consider whether to import known luminescence systems with
common synthetic biology chassis cells.

## 3.Design and Build:

In order to determine the final chassis cell to be used, we
investigated a variety of luminescent bacteria, analysed the principles of
bioluminescence, and took chassis cells commonly used in synthetic biology into
consideration.

**Table. 1 | Comparison of bacterial project adaptability**

|  | Vibrio fischeri | Vibrio qinghaiensis | Vibrio harveyi | E. coli | Vibrio natriegens |
| --- | --- | --- | --- | --- | --- |
| advantages | Self-luminous; Tolerant of the sea; non-pathogenic | Self-luminous; non-pathogenic | Self-luminous; marine | More commonly used chassis cell; easy to operate | Rapid growth; Tolerant of the sea; non-pathogenic |
| drawbacks | Luminescence dependent Quorum sensing | Freshwater luminous bacteria; not adapted to the sea | virulence | Not adapted to the sea; non-self-illuminating | non-self-illuminating |

All luminescent bacteria have biochemically similar luminescence systems, all of
which are luciferase luminescent encoded by a cluster of lux genes, and the reaction
mechanism involves the oxidation of flavin mononucleotide (FMNH2) and long-chain
aliphatic aldehydes (RCHO) in the reduced state to FMN and long-chain fatty acids
(RCOOH) in the presence of molecular oxygen and luciferase and the emission of light
with a wavelength of 490 nm of blue-green light.Bacterial luciferase consists of a
heterodimer containing an α-subunit (molecular weight 42 kDa) and a β-subunit
(molecular weight 37 kDa), and is active only when the two subunits coexist. The
optimal temperature for this enzyme reaction is 18°C, and it is rapidly inactivated
above 25°C. This luminescence system has been introduced into the chassis cell by
mature means, so we chose this as the basic luminescence line.

## 4.Test:

Firstly, we tested the autoluminescent organisms first, since Vibrio qinghaiensis and
Vibrio harveyi were obviously not compatible with the requirements, we cultured
Vibrio fischeri in the laboratory for the test (culture conditions: 22℃ 16h static
cultivation), and successfully observed the bioluminescence phenomenon.

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-16.png)

By analysing the results of the experiments, we found that Vibrio fischeri can emit
fluorescence visible to the naked eye, which can indicate the effectiveness of the
luminescence system. However, we also found that the growth rate of Vibrio fischeri
is too slow and its luminescence is dependent on Quorum sensing, which does not meet
the requirement of rapid proliferation and luminescence.

Under the guidance of PI, we discovered a new fast-growing chassis cell developed in
recent years: Vibrio natriegens, which is the shortest known free-living bacterium,
with a generation time of less than 10 min, and is a more mature synthetic biology
tool than Vibrio fischeri.synthetic biology tool, and is better suited for
subsequent lineage transformations.

## 5.Learn:

We ultimately chose to use Vibrio natriegens as the chassis cell and plan to
introduce the lux gene encoding luciferase to enable it to bioluminesce. In this
way, the engineered bacteria will be able to utilise their properties to meet the
requirements of the rescue standard. After selecting the appropriate strain, at this
point, due to the uncertainty of the environmental factors, we considered the degree
of adaptation of the engineered bacteria to the external environment in more depth.
In order to ensure the effectiveness of the product, we need to explore ways to
enhance the adaptability of the engineered bacteria in order to improve the
survivability.

# Cycle2: Microbial Survival

## 1.Research:

We first learnt that the average temperature of the world's oceans is around 20°C,
with lower temperatures in most seas. Through reviewing the literature, we found
that although Vibrio natriegens is a bacterium of marine origin, its optimal growth
temperature is 37℃. We believe that the low temperature stress in the ocean has a
great impact on the effectiveness of the engineered bacteria, and that low
temperatures can lead to ROS accumulation inducing cell death. After research we
identified two areas that need to be targeted for a solution: metabolic
replenishment and elimination of ROS accumulation. In addition, when the application
scenario is transferred to the space, the engineered bacteria not only face the
challenge of low temperature, but also the threat of radiation to their survival.
Our famous synthetic biologist, Prof. Xianen Zhang, confirmed our consideration of
radiation in space, and he believed that the simple use of physical isolation is
costly and not conducive to the maintenance of the activity of the algal system. He
suggested that we should learn about Deinococcus radiodurans, understand its defence
mechanism and apply the core function part flexibly to our chassis cell.

## 2.Imagine:

Firstly, we want to reduce the metabolic impact of low temperature stress on Vibrio
natriegens. We thought of supporting the survival of Vibrio natriegens by providing
additional efficient carbon source, which can provide more metabolic materials for
Vibrio natriegens to reduce the impact. Secondly, low temperature can coerce the
bacteria to accumulate ROS in a stress response and lead to cell death, and we want
to reduce the accumulation of ROS. We thought of achieving this by inducing the
activation of the expression of enzymes involved in ROS degradation. Can we fuse
these two solutions to be embodied in fewer gene lines to reduce the metabolic
burden on the bacteria? And for defence against space radiation, can we rationalise
the expression of key proteins that play a role in radiation protection to confer
radiation resistance to engineered bacteria?

## 3.Design and Build:

We found an algal-bacterial coexistence system through literature
search. One of them, Polychlorophyta PCC7942, imported the cscB sucrose permease
gene and expressed a sucrose/proton isotransporter capable of secreting sucrose. The
alga is able to utilise sunlight and CO2 produced by Vibrio natriegens'
life activities for photosynthetic carbon cycling, generating sucrose which is then
secreted extracellularly with the aid of the transporter, and Vibrio natriegens is
able to utilise sucrose as a carbon source for metabolism (see Figure. A)[8]. In order to enhance the salt tolerance of Polycystis
aeruginosa, we selected a salt-tolerant strain of PCC7002. This algal coexistence
system solved the metabolic aspects and we built on this system for cold-tolerant
lines. We learned that ROS are converted to H2O2 by Superoxide dismutase
(SOD), and then catalase catalyses the decomposition to H2O and
O2 . We then established a sucrose-sensing switch in Vibrio natriegens,
pSacB , which can be activated in the presence of sucrose to express the
downstream SOD, catalase to catabolise ROS. In addition, we have learnt that RecF
and RecO of Deinococcus radiodurans have been applied to the construction of
prokaryotic expression systems to confer radiation resistance to prokaryotes
(see Figure. B). In this way, the microbial survival part of the
problem is solved.

![Gut-sweeper](https://static.igem.wiki/teams/5210/result-23.jpg)

## 4.Test:

We determined the molecular weight of each protein by querying. Since the effect of
pSacB promoter is more clear, our experiment mainly explored whether SOD
and catalase can be expressed.Pet-28a was used as a vector for two target genes, and
the proteins were expressed in engineering bacteria by IPTG induction. We set up two
groups of 0 mM IPTG treatment and 0.5 mM IPTG treatment according to the
pre-experiment. SDS-PAGE experiments were carried out after low-temperature
induction to initially observe the expression of target proteins (see Figure
A). As for exploring the expression of RecO and RecF, we introduced the
two genes into Pet-30b vector separately as two experimental groups, and induced
them with 0.5 mM IPTG for 1h, 2h, 3h and 4h, respectively, and the same
low-temperature induction was carried out to perform SDS-PAGE experiments, and the
two groups were compared for observing the expression of the target proteins
(see Figure B).

![Gut-sweeper](https://static.igem.wiki/teams/5210/result-24.jpg)

In addition, we tested the anti-radiation effect of RecO and RecF, with untransformed
and blank plasmid-transformed E. coli as control groups.

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-19.png)

## 5.Learn:

In this cycle we have addressed the issue of the impact of environmental variables on
the survival of microorganisms. However, a new problem arose: the product is not in
constant use in specialised scenarios, such as rescue at sea, and should normally be
"asleep". At the same time, we believe that the product needs to be well preserved
and quickly recovered.

# Cycle3: Product Preservation

## 1.Research:

We have conducted a preliminary exploration of sea rescue as an application scenario
for our product. The luminescence needs to be limited by the ocean scenario, i.e.,
luminescence only occurs in the ocean. The high osmotic environment of the ocean can
be used as a prerequisite for luminescence. In addition, our products are dormant in
normal times and only function when needed. Day-to-day preservation of our products
is what we want to focus on in this cycle.

## 2.Imagine:

In the marine application scenario, can genetic elements be introduced to feel the
luminescence of hypertonic environment? To avoid accidental activation of the
product when it is not in daily use state, is there any preservation method to make
the engineered bacteria temporarily dormant and revive them when in use?

## 3.Design and Build:

For sensing the hypertonic environment, we used the Envz-OmpR
two-component system. The effectiveness of this system has been described in detail
in the luminescence section of the engineer cycle. As for the preservation method,
we looked up the following commonly used bacterial preservation methods to choose
from.

**Table. 2 |**
**Comparison of bacterial preservation methods**

|  | Cryopreservation | Lyophilised preservation | Agar slant culture | Oil Seal Preservation |
| --- | --- | --- | --- | --- |
| vantage | Can be stored for several years, suitable for most bacteria | Long shelf life, good stability, easy to transport, no need for continuous energy supply | Simple method, low cost | Can be stored at room temperature without special equipment |
| drawbacks | Requires special equipment and is costly | The freeze-drying process may result in the death of a portion of the bacteria | Requires medium | Relatively short shelf life and need for medium |

Considering the stability, convenience, commerciality and long-term effectiveness of
the preservation method, we finally choose the lyophilised powder preservation
method. (ps: Although it will cause the death of some bacteria, our engineered
bacteria grow fast, so as long as we ensure that there are bacteria surviving, it
will not affect the effectiveness of the product too much.)

## 4.Test:

Lyophilised powder preservation technology is more mature nowadays, and the purpose
of our test is mainly to determine whether the lyophilised powder technology can be
applied to our products. We resuspended the bacterial particles in 10% sucrose LB
medium and subjected them to a gradient freezing treatment, with the treatment
temperatures being successively: -4°C, -20°C, and -80°C. The powder was then made
into lyophilised powder using a lyophiliser (see Figure A). Then the lyophilised
bacterial powder was made using a lyophiliser (see Figure A). Then
we added LB medium to the ep tube containing the lyophilised bacterial powder for
resuscitation to dissolve it completely. After that, we performed operations such as
plate coating and bacterial inoculation to consider the bacterial growth(see
Figures B, C, and D), and found that the growth state of the
lyophilised bacterial powder was close to the normal level after resuscitation.

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-20.png)

## 5.Learn:

The testing section illustrates the ability of our product to use lyophilised powder
as a form of bacterial preservation. At this point we have probably perfected a
series of safety issues before the product can be used.

# Cycle4: Preventing false positives and leakage

## 1.Research:

Our synthetic biologist, Prof He Jin, has raised the issue of false positives on the
safety of our products, that is, the situation where the luminescent bacteria drift
and are not localised properly. In response to this problem, our stem group used a
filter membrane and electrostatic spinning technology to ensure that the luminescent
bacteria were positioned around the target. After further consideration of Professor
He Jin's suggestion, we found that the problem of false positives cannot be fully
solved by hardware, but also by biological leakage caused by hardware damage. In
addition, biological leakage can also cause a series of ecological safety problems.
Therefore, in this cycle, we mainly address the safety issues caused by microbial
leakage.

## 2.Imagine:

The previous construction of the algae-bacteria coexistence system and the
cold-resistant module can allow the leaking engineering bacteria to passively die
due to the lack of carbon source and the accumulation of ROS. However, other
scenarios are not necessarily low carbon source and low temperature. Considering the
future multi-scenario applicability of the product, can we engineer bacteria to be
able to actively commit suicide after leakage?

## 3.Design and Build:

We designed two lines to sense sucrose concentration to achieve
active suicide. Firstly, we used PSacB to sense sucrose concentration,
and then set up the repressor protein lacI to control the expression of MazF toxin
protein. Line 1 reduces the secretion of lacI when sucrose concentration decreases
around the engineered bacteria, thus weakening the deterrence of Plac by
lacI, which in turn expresses MazF to kill the bacteria. Considering the expression
level of PSacB and the possibility of killing when it is not used. We
improved the sensing of sucrose according to line I. Line II enabled the engineered
bacteria to activate the sensing of sucrose only in the hypertonic environment of
the ocean, and after entering the ocean, the spare sucrose in the product and the
sucrose produced by the algae inhibited the deterrence of Pscr by ScrR,
which led to the expression of lacI and the non-expression of MazF. After the
engineered bacteria were detached from the system, the sucrose concentration was
reduced, the inhibition of Pscr by ScrR was enhanced, the expression of
lacI was reduced, and MazF was then expressed to play a killing role.

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-21.png)

## 4.Test:

We mainly test the line two part. Due to the complexity of the line, this part of the
test we would like to analyse the repressor protein in the line by molecular docking
technique. lacI is a repressor protein commonly used in the field of synthetic
biology, and its deterrent effect on lac is relatively precise.
Therefore, in order to verify the validity of sucrose sensing, we talked about
docking ScrR and scr to analyse the binding of both before sucrose
treatment.

After analysis, we found that the conformation of ScrR protein did change after MD
simulation of sucrose treatment (see Figure. A), and the affinity of
ScrR to Plac before and after simulation did not change significantly,
but the binding mode changed. The results showed that only a small portion of
sucrose-treated ScrR binds to Plac , and the DNA position of
Plac is far away from ScrR, which allows space for the transcriptase to
bind to Plac to activate transcription (see Figures B, C, and
D).

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-22.png)

## 5.Learn:

We have constructed an active kill line that senses changes in sucrose concentration,
forming a comprehensive passive-active anti-leakage module. In this way, our
products are largely protected against problems such as false positives and
ecological damage.

# Cycle5: Product recovery

## 1.Research:

We addressed the safety of product use in the previous cycle, and this cycle needed
to address the safety of the product after use to ensure the safety of the entire
product line. We focused on brainstorming ways to deal with microorganisms after
use.

## 2.Imagine:

Can we improve on the methodology of previous cycles to get a treatment plan?

## 3.Design and Build:

We have a two-way approach to this.

①The first is the conservative treatment of artificially induced killing: we
initially designed a temperature control system and a drug control system to promote
engineered bacterial toxin proteins to trigger bacterial death by controlling the
temperature or adding inducers.

![Gut-sweeper](https://static.igem.wiki/teams/5210/engineer-cycle/engineer-cycle-23.png)

②Secondly, there is the recycling method of freeze-dried recovery: our products are
preserved in the form of freeze-dried powder, and the recycling of products can be
achieved by reformulating the used products into freeze-dried powder. Of course,
recycling is an idea and may need to be put into practice to prove.

## 4.Test:

We mainly reviewed and screened the drug control system. We initially selected two
commonly used inducers: arabinose and rhamnose, however, when comparing the
properties of these two inducers, we found that rhamnose degrades slowly in the
natural environment and may remain in the water column for a long period of time,
causing potential bioaccumulation and environmental pollution. Rhamnose may have
negative effects on aquatic organisms, especially microorganisms and aquatic plants.
High concentrations of rhamnose may interfere with the physiological and ecosystem
functions of aquatic organisms. Compared to rhamnose, arabinose is generally
considered to have less or negligible impacts in the marine environment. Arabinose
is a naturally occurring simple carbohydrate that can be utilised as a carbon source
by many micro-organisms and aquatic organisms. It is usually well biodegradable and
can be rapidly broken down and utilised by microorganisms. It is a natural product
that does not usually cause discomfort or toxic reactions in organisms, is usually
released in small amounts in the marine environment and can be rapidly broken down
by micro-organisms in the water without causing long-term accumulation or pollution
problems.

## 5.Learn:

Therefore, we considered arabinose as a better choice to construct a simple
drug-inducing system. However, whether the inducer can effectively kill the
engineered bacteria in seawater still needs further experimental proof. In this
cycle we mainly propose solutions for the safety of the product after use, and the
test results can only initially reflect the feasibility of these constructive
methods, after which more rigorous proof is needed.

# References

1. Brodl, E. et al., The impact of LuxF on light intensity in bacterial
   bioluminescence. j Photochem Photobiol B **207** 111881 (2020).
2. Nijvipakul, S. et al., LuxG is a functioning flavin reductase for
   bacterial luminescence. j BACTERIOL **190**1531 (2008).
3. Kenney, L. J. & Anand, G. S., EnvZ/OmpR Two-Component Signaling: an
   Archetype System That Can Function Noncanonically. ecoSal Plus **9**
   (2020).
4. Smith, B. T. & Walker, G. C., Mutagenesis and more: umuDC and the
   Escherichia coli SOS response. genetics **148** 1599 (1998).
5. Ye HF and Niu LX and Qiao LL and Wang ZH and Guan NZ. A prokaryotic
   far-red light-regulated transcriptional activation device and its
   construction method and application in tumour therapy. CN Patent
   202210607219.2. Dec 08, 2023.
6. Visick, K. L., Stabb, E. V. & Ruby, E. G., A lasting symbiosis: how
   Vibrio fischeri finds a squid partner and persists within its natural host.
   NAT rev microbiol **19** 654 (2021).
7. Mandel, M. J. et al., Squid-derived chitin oligosaccharides are a
   chemotactic signal during colonisation by Vibrio fischeri. Appl Environ
   Microbiol **78** 4620 (2012).
8. Li, C. et al., A Highly Compatible Phototrophic Community for
   Carbon-Negative Biosynthesis. Angew Chem Int Ed Engl **62** e202215013
   (2023)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Experiments.{% endblock %}
{% block page\_content %}

### Experiments

Scroll To Explore

![]()

Protocols

Experiment results

# 1. Configuration of LB medium

Prepare 200 ml of medium by weighing 2 g of tryptone, 1 g of yeast extract, 2 g of NaCl, and 3 g of agar powder, adding 200 ml of purified water, and stirring with a glass rod until the solutes are dissolved. The pH was adjusted to 7.0-7.4 with 5 mol/L NaOH (usually 40 μl), and then the mixture was dispensed into 250 ml conical flasks. The conical flask was sealed with a sealing film and secured with a rubber band. Then steam sterilize the mixture under high pressure at 121°C, 103.4 kPa for 20 min. remove it, place it on an ultra-clean bench, cool it down to 50~55°C, and then add the antibiotic (kanamycin) to a final working concentration of 50 μg/ml.

# 2. PCR amplification

Take the ice box and put dNTP, PrimeSTAR enzyme, 5X PrimeSTAR buffer, upstream and downstream primers, template plasmid, and dd water, into the ice box, and leave it to melt. Take the corresponding number of microtubes and place them in the ice box. Configure the reaction system according to primestar. Note the amount of template used (concentration determines volume)

| Reaction System Composition | Spiked volume | Final concentration |
| --- | --- | --- |
| 5 X PrimeSTAR Buffer (Mg2+Plus) | 10μl | 1 X |
| dNTP Mixture (2.5mM each) | 4μl | 200μM each || Primer1 | 10-15pmol | 0.2-0.3μM |
| Primer2 | 10-15pmol | 0.2-0.3μM |
| Template | <200ng |  |
| PrimeSTAR HS DNA Polymerase (2.5U/μl) | 0.5ul | 1.25units/50μl |
| DdH2O | Up to 50 μl |  |

Note: The total volume is 50 μL

Turn on the PCR instrument and set up the program according to the reaction conditions.

# 3. Electrophoretic verification of PCR results

Preparation of gel: weigh 0.35g agar powder, add it into 30ml 1X TAE (Tris-acetic acid electrophoresis buffer) flask, microwave heating for 2.5min, and cool it down to 50 ℃. Remove the gel from the 4 ℃ refrigerator and aspirate 3 ml with a special gun for electrophoresis, stick the gun tip under the liquid surface and rotate it slightly to mix it well. Pour the liquid into the grid for dispensing the gel, insert the comb and wait for solidification. Sample Preparation: After PCR is finished, remove each reaction tube, take 5μl of each and add it to a new microtube, label it, add 1ul of 6X Sampling Buffer to each and mix well.

Run the gel: take out the Marker from -20℃ refrigerator, pull out the comb from the solidified gel, put it into the electrophoresis tank according to the direction, fill with 1×TAE add the prepared samples into each gel well and add 5ul of melted Marker, record the order of the samples in the wells. Run the gel at 150V constant pressure. Observe under UV light and compare the position of the fluorescent bands with the labeling instructions. Take photos.

# 4. DNA purification and recovery

We adopt the faster column purification and recovery method, and use the universal DNA purification and recovery kit (DP214) from TIANGEN Biochemical Technology, which mainly recovers DNA from agarose gel.

**Cut the gel:** use a clean blade to cut off the target DNA bands, try not to cut more than one, put it into a 1.5 mL ep tube (cut the gel quickly, avoid UV light), weigh the total weight and the weight of the empty 1.5 ml ep tube to calculate the weight of the gel block.

**Solubilization:** Add an equal volume of solution PC to the gel block for solubilization (100 μl of PC solution for every 0.1 g of gel block), and water bath at 50℃ for 10 min, during which the centrifuge tube is constantly and gently turned up and down to ensure that the gel block is completely solubilized.

**Column equilibrium:** put the adsorption column CB2 of the kit into the collection tube, add 500 μl of equilibrium solution BL, centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and adsorption column CB2 into the collection tube.

**Adsorption:** Add the solution obtained from the lysate to the adsorption column CB2 in the collection tube, centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.

**Wash:** Add 600 μl of rinse solution PW to the adsorbent column CB2, centrifuge at 12,000 rpm for 1 min, pour out the waste liquid from the collection tube, and put the adsorbent column CB2 into the collection tube. If high purity is required, the column should be washed again with the same procedure.

**Drying:** The column CB2 is centrifuged at 12,000 rpm for 2 min to remove as much of the rinse solution as possible. Leave the column at room temperature for a few minutes and then dry thoroughly. (The rinse solution contains ethanol, and ethanol residues can interfere with subsequent enzyme reaction experiments.)

**Elution:** Place the adsorbent column CB2 into a clean centrifuge tube and add an appropriate amount of elution buffer EB to the center of the adsorbent membrane (if the recovered target fragment is >4kb, the elution buffer EB should be heated in a water bath at 65-70℃) and leave it at room temperature for 2 min. Centrifuge the adsorbent column at 12,000rpm for 2 min and collect the DNA solution. After measuring the DNA concentration, store the DNA solution at -20℃.

# 5. Enzymatic digestion of PCR products

Use Thermo Fisher Scientific's FastDigest for enzyme digestion, take EcoR Ⅰ and Hind Ⅲ enzyme digestion sites for example：

![Gut-sweeper](https://static.igem.wiki/teams/5210/protocol-5.png)

Our enzymatic system is presented below:

| Reaction System Composition | Spiked volume |
| --- | --- |
| DdH2O | 24μl |
| FastDigest enzyme(EcoR Ⅰ) | 1μl |
| FastDigest enzyme(Hind Ⅲ) | 1μl |
| 10×FastDigest Buffer   Or 10×FastDigest Green Buffer | 4μl |
| DNA(Plasmid DNA/PCR product) | 2μl/10μl |
| Total volume | 32μl/40μl |

Incubate at 37℃ in water bath for 15min, heat at 80℃ for 10min to terminate the enzyme digestion, recover the DNA product.

# 6. Plasmid introduction of fragment carriers

The appropriate molar ratio of plasmid vector to target gene fragment is 1:4 to 1:3, while the required volume ratio is calculated based on concentration and fragment length.

The reaction needs to be carried out on ice and the reaction system is as follows:

| Reaction System Composition | Spiked volume |
| --- | --- |
| Plasmid gel recovery products | 2μl |
| PCR digestion of recovered products | 8μl |

# 7. Plasmid extraction:TIANGEN Plasmid Extraction Kit (DP103)

1. Place the adsorption column CP3 into the collection tube, add 500 μl of equilibrium solution BL to the adsorption column, centrifuge at 12,000 rpm for 1 min, pour out the waste liquid from the collection tube, and place the adsorption column back into the collection tube.

2. Take 1-5 ml of the overnight culture, add it to the centrifuge tube, centrifuge at 12,000 rpm for 1 min using a conventional tabletop centrifuge, and aspirate the supernatant as much as possible.

3. Add 150 μl of Solution P1 (with RNaseA added) to the centrifuge tube with the bacterial precipitate and thoroughly suspend the bacterial precipitate using a pipette or vortex shaker.

4. Add 150 μl of Solution P2 to the centrifuge tube and gently spin up and down 6-8 times to completely lyse the organism. Due to the use of TIANRed, the color of the solution will be a clear purple when Solution P2 is added and mixed thoroughly. If there is a mixture of cloudy red and purple colors, the lysis is not complete. Continue mixing until the color of the solution is completely clear purple.

5. Add 350 μl of Solution P5 to a centrifuge tube and immediately mix rapidly up and down 12-20 times; when well mixed, a flocculated precipitate will appear. Centrifuge at 12,000 rpm for 2 min.

6. Transfer the supernatant collected in the previous step to the adsorbent column CP3 using a pipette (the column is placed in the collection tube), taking care not to aspirate the precipitate as much as possible. Centrifuge the column at 12,000 rpm for 30 s, pour out the waste liquid from the collection tube, and then place the column CP3 into the collection tube.

7. Add 300 μl of rinse solution PWT to the adsorbent column CP3, centrifuge at 12,000 rpm for 30 s, and pour out the waste solution in the collection tube.

8. Place the adsorption column CP3 into the collection tube and centrifuge it at 12,000 rpm for 1 min to remove the rinse solution remaining on the adsorption column.

9. Place the adsorbent column CP3 in a clean centrifuge tube, add 50-100 μl of elution buffer TB dropwise to the middle of the adsorbent membrane, centrifuge at 12,000 rpm for 30 s, and collect the plasmid solution into the centrifuge tube.

# 8. Plasmid transformation coating and inoculation

**Heat-excited transformation:**Make a sufficient amount of ice into a foam box as an ice bath, take the plasmid, receptive ep tubes containing 50 μL of E. coli and place them on the ice. Add 5μl of plasmid to the receptor ep tube and leave it on ice for 30 min. start the water bath and set the temperature to 42°C. Take out the receptor ep tube from the ice bath and heat stress it. Remove the sensory ep tubes from the ice bath, heat-stimulate for 60 to 90 s, and immediately place back on ice to cool. after 2 min, add 500 μl of liquid LB.

**Coating plate:** After the addition of LB, the receptor ep tubes were placed on a suspension plate and shaken in a shaker shaker at 37°C, 180 rpm, for 40 min. and then centrifuged in a centrifuge at 6000 rpm for 1 min. Take the sensory ep tube and the screening plate containing antibiotics, pipette gun on the ultra-clean bench, aspirate and discard 500 μl of the liquid in the sensory ep tube, the remaining liquid was blown with a pipette gun and then carefully poured into the LB Petri dish, the liquid was evenly coated with a blotting rod and the dish was capped, and then inverted in the incubator overnight. Observe the bacterial growth status on the next day, and if the growth status is good, put it into 4℃ refrigerator for storage.

**Bacterial inoculation:** Take the LB liquid medium containing corresponding antibiotics (5ml per tube) and the bacterial plate in 4℃ refrigerator into the ultra-clean table, take metal tweezers, fire-roasted and sterilized, clip a small gun tip, zap it into a single colony of LB petri dish to pick bacteria, and put it into the LB liquid medium vertically. Put the small test tube with culture medium into the shaker for incubation, 37℃, 180 rpm.

**Points of Attention:** Avoid touching the bottom of the sensory ep tube with hands as much as possible during the whole process; the empty LB petri dish needs to be put in the incubator at 37℃ for drying if there is moisture before picking up the bacteria; the use of ultra-clean table should be sterilized 30min in advance.

# 9. Expansion and IPTG induction

**Expansion of bacteria:**Prepare 500 ml of LB in a conical flask sterilizer and sterilize it, after cooling, add the corresponding concentration of antibiotics, take the LB medium that was incubated overnight with the bacteria, and pour 5 ml of the bacterial solution. Seal the conical flask and fix it with rubber band. Place the conical flask in a shaker, incubate at 37℃ 180 rpm until the OD value is between 0.6 and 0.8 (turbid to the point that you can't see your fingers through the bacterial liquid). Activate the shaker refrigeration key to reduce the temperature of the shaker thermostatic oscillator to 10℃.

**IPTG induction:**Add IPTG mother liquor in a conical flask to make the system IPTG concentration of 0.5 mM, put it into the shaker constant temperature oscillator at 10℃, and take it out for observation after 16h.

# 10. Validation of the construction of osmotic pressure gradient for LB medium in two-component system

### (1) Pre-experiment: Determination of the appropriate osmolality range:

|  | Tryptone | Yeast extract | Purified water | NaCl |
| --- | --- | --- | --- | --- |
| 1M | 2g | 1g | 200ml | 11.68g |
| 2M | 2g | 1g | 200ml | 23.36g |
| 3M | 2g | 1g | 200ml | 35.04g |
| 4M | 2g | 1g | 200ml | 46.72g |
| 5M | 2g | 1g | 200ml | 58.40g |

### (2) Formal experiment: validation of hyperosmotic activation of two-component system:

|  | Tryptone | Yeast extract | Purified water | NaCl |
| --- | --- | --- | --- | --- |
| 0M | 2g | 1g | 200ml | 0g |
| 0.17M (Normal LB) | 2g | 1g | 200ml | 2.00g |
| 0.25M | 2g | 1g | 200ml | 2.92g |
| 0.5M | 2g | 1g | 200ml | 5.84g |
| 0.75M | 2g | 1g | 200ml | 8.76g |
| 1M | 2g | 1g | 200ml | 11.68g |

# 11. Growth curve measuring by enzyme-labeled instrument

## (1) Pretreatment of 96-well plates and lids

1. prepare 10% Triton X-100 in ethanol by mixing 1 ml of Triton X-100 with 9 ml of ethanol.

2. Pour 10 ml of diluted Triton X-100 (10% Triton X-100) into a new microplate lid.

3. Incubate the lid with diluted Triton X-100 for 15 s at room temperature.

4. Take a new 96-well plate and fill each well with 100% ethanol (400 μL per well). Incubate at room temperature for 15min
incubate at room temperature for 15min.

5. Discard ethanol from the wells of the microplate and discard Triton X-100 from the microplate lid.

6. Air-dry the wells and lids in an ultra-clean bench for 30 min.

7. Turn on the ultra-clean UV lamp to further sterilize the microplate wells and lids for 15min.

## (2) Sample test

| Plasmids | Sodium butyrate(mM) | Bacteria solution(μl) | Total volume(μl) | Replicates | Resistance |
| --- | --- | --- | --- | --- | --- |
| [pet28a]-Pompc-eGFP | 0 | 5 | 200 | 3 | KN |
| [pet28a]-Pompc-eGFP | 0.17 | 5 | 200 | 3 | KN |
| [pet28a]-Pompc-eGFP | 0.25 | 5 | 200 | 3 | KN |
| [pet28a]-Pompc-eGFP | 0.5 | 5 | 200 | 3 | KN |
| [pet28a]-Pompc-eGFP | 0.75 | 5 | 200 | 3 | KN |
| [pet28a]-Pompc-eGFP | 1 | 5 | 200 | 3 | KN |

## (3) Enzyme marker and software settings

1. Turn on the microplate reader and open the Gen5 software.

2. Adjust the parameters and measure the bacterial growth curve.

3. Plate type: 96-well plate. b. Lid use: Check.

4. Lid use: checked.

5. Temperature: 37°C.

6. Shake: Track 0:10.

7. Total time required (e.g., 16 hours, 24 hours, or 48 hours) and kinetic measurements at 10-minute intervals with continuous orbital shaking.

8. Readings: Absorption at 600 nm.

## (4) Organize the results

1. Select the data type as OD600 to observe the results.

2. After completing data collection, remove the 96-well plate from the microplate reader.

3. Sterilize the sample and discard the microplate properly.

4. save the results in the Gen5 software and export the data to an Excel spreadsheet file. 5. close the Gen5 software and export the data to an Excel spreadsheet file.

# 12. Fluorescence and luminescence intensity measuring by enzyme-labeled instrument

## (1) Pretreatment of 96-well plates and lids

1. prepare 10% Triton X-100 in ethanol by mixing 1 ml of Triton X-100 with 9 ml of ethanol.

2. Pour 10 ml of diluted Triton X-100 (10% Triton X-100) into a new microplate lid.

3. Incubate the lid with diluted Triton X-100 for 15 s at room temperature.

4. Take a new 96-well plate and fill each well with 100% ethanol (400 μL per well). Incubate at room temperature for 15min
incubate at room temperature for 15min.

5. Discard ethanol from the wells of the microplate and discard Triton X-100 from the microplate lid.

6. Air-dry the wells and lids in an ultra-clean bench for 30 min.

7. Turn on the ultra-clean UV lamp to further sterilize the microplate wells and lids for 15min.

## (2) Sample test

1. Add 200μL of the sample to be tested into the wells, and set up 3 replicate wells for each sample.

2. Add 200μL of blank LB to the blank wells as a blank control.

## (3) Enzyme marker and software settings

1. Turn on the microplate reader and open the Gen5 software.

2. Adjust the parameters and measure the bacterial growth curve.

3. Plate type: 96-well plate. b. Lid use: Check.

4. Lid use: checked.

5. Temperature: 37°C.

6. Shake: Track 0:10.

7. Total time required (e.g., 16 hours, 24 hours, or 48 hours) and kinetic measurements at 10-minute intervals with continuous orbital shaking.

8. Readings: Absorption at 600 nm；luminescence at 490nm.

## (4) Organize the results

1. Select the data type as OD600 and luminescence to observe the results.

2. After completing data collection, remove the 96-well plate from the microplate reader.

3. Sterilize the sample and discard the microplate properly.

4. save the results in the Gen5 software and export the data to an Excel spreadsheet file. 5. close the Gen5 software and export the data to an Excel spreadsheet file.

# 13. SDS-PAGE

**Protein sample preparation:**
After shaking the amplified bacterial solution well, take 1mL in a centrifuge tube and centrifuge at 10,000 rpm for 10 min. remove the supernatant, leaving about 50ul or so, add a small amount of pH 7.4 PBS to wash three times, leaving about 50ul. Add 50uL 1×Loading Buffer, 0.31g DTT and shake to suspend the precipitate. Place the centrifuge tube in a water bath at 100°C for 10 min. centrifuge at 12,000 rpm for 5-10 min, and the supernatant is the extracted protein. Transfer the supernatant to a new centrifuge tube and add a small amount of Loading Buffer (about 5ul) for sampling.

**SDS-PAGE gel electrophoresis:**
After shaking the amplified bacterial solution well, take 1mL in a centrifuge tube and centrifuge at 10,000 rpm for 10 min. remove the supernatant, leaving about 50ul or so, add a small amount of pH 7.4 PBS to wash three times, leaving about 50ul. Add 50uL 1×Loading Buffer, 0.31g DTT and shake to suspend the precipitate. Place the centrifuge tube in a water bath at 100°C for 10 min. centrifuge at 12,000 rpm for 5-10 min, and the supernatant is the extracted protein. Transfer the supernatant to a new centrifuge tube and add a small amount of Loading Buffer (about 5ul) for sampling.

**Staining and decolorization:**
After electrophoresis, recycle the electrophoresis solution, pry open the glass plate, remove the gel into the box and spread it flat. Pour the Caulmers Brilliant Blue staining solution into the cassette, remove it after microwave heating for 2min, and decolorize it with ethanol-acetic acid decolorizing solution, and change the decolorizing solution every 30min until the bands were clearly seen. Pour off the decolorizing solution and observe the bands under a bright background.

# 14. Preparation and recovery of freeze-dried bacterial powder

1. incubate overnight on LB medium at 37°C with shaking (OD≈1-1.5)

2. Centrifuge 1 ml of bacterial suspension (12000r, 2 min) and discard supernatant.

3. Resuspend the bacterial pellet in 500µL of 10% sucrose LB medium.

4. Transfer to EP tubes and seal with sealing film (holes punched in film to equalize air pressure).

5. Gradient freezing pretreatment: -4℃ for 30min, -20℃ for 30min, -80℃ for 1h.

6. Start the lyophilizer to pre-freeze for 2 hours, let the chamber temperature drop to -50°C

7. Sublimate under vacuum for 12 hours

8. Increase the temperature to about 30°C to desorb the residual water under vacuum. The lyophilized powder can be stored at -20°C for a long time.

9. Resuscitation: add 1ml of LB medium into the EP tube to dissolve the powder completely.

10. Determine the growth curve of resuscitated bacterial powder by using microplate reader.

# 15. RecO, RecF anti-UV radiation damage effect verification experiment

## (1)Colony plate counting

1. Single colonies of recO and recF positive strains and single colonies of E.coli BL21(DE3) containing the pET30b null plasmid and wild-type were picked and incubated overnight at a final concentration of 50 μg/ml kana 5 ml LB culture medium at 37℃, 180 r/min with shaking bed shaking.

2. Remove the four strains of the overnight culture from the shaker, and dispense them into four autoclaved 15 ml centrifuge tubes, centrifuge for 3 min at 8,000 r/min at room temperature, and discard the supernatant. After adding 10 ml of 0.01M PBS to resuspend the precipitate, centrifuge again at 8,000 r/min for 3 min at room temperature and discard the supernatant.

3. Add 10 ml PBS to adjust the OD600 of each bacterium close to 1 (spectrophotometer or microplate reader detection)

4. Take 6 sterile test tubes, numbered 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, take 7 LB solid medium for each strain of bacteria, numbered 10-5, 10-6, and make 3 duplicate plates for each dilution concentration, and leave one plate as blank control. Pipette 4.5 ml of sterile saline into the above numbered tubes with a 5 ml pipette in aseptic operation.

5. Shake the bacterial solution well, then use a 1 ml sterile pipette to blow back and forth several times in the original sample to be diluted, and then accurately pipette 0.5 ml of bacterial solution into the test tube labeled 10-1. Take another 1 ml sterile pipette, in the same way, first blow the sample back and forth several times in the test tube labeled 10-1 and accurately pipette 0.5 ml of the bacterial solution into the test tube labeled 10-2, and so on until the dilution in the test tube labeled 10-6.

6. Use 1 ml sterile pipette to suck 0.1 ml of 10-5 and 10-6 diluted bacterial solution accurately, add it to the corresponding numbered LB solid medium, spread it evenly with a pasteurized tube, and put it into 37℃ constant temperature incubator for incubation.

7. Remove the plates after 24 h of incubation, select the petri dishes with colony numbers within the range of 3-300/plate, calculate the number of colonies on each plate, and calculate the results according to the formula (the number of viable bacteria (pcs/ml) = [(χ1 + χ2 + χ3)/3] × 10 × the number of dilutions).

## (2)Effect of UV light on bacterial survival

1. Take the bacterial solution with adjusted OD and counted, 12,000 r/min, centrifuge for 2 min, discard the supernatant. Resuspend the bacterial solution in 1 ml PBS, and mix the bacteria with sufficient shaking.

2. Preheat the UV lamp; adjust the irradiation power of the UV lamp and the irradiation table to 1 J/s, and turn on the UV lamp to preheat for 30 min before irradiation to stabilize the intensity of the UV lamp.

3. For each strain of bacteria, take 25 pieces of LB solid culture medium (so that the final concentration of IPTG in the medium is 0.1 mM), add 0.1 ml of bacterial solution to each medium, and spread it evenly on the surface of the medium with an L stick. The UV irradiation dose (25 J/m2, 50 J/m2, 75 J/m2, 100 J/m2, 125 J/m2, 150 J/m2, 175 J/m2, 200 J/m2) was labeled on the surface of the culture plate, and three replicate plates were made for each irradiation dose, and one plate was left as a blank control.

4. Start irradiation according to the irradiation dose marked on each plate. After irradiation, all the culture plates were wrapped with black paper, inverted and placed in 37℃ constant temperature incubator, and then taken out and counted on the 3rd day.

# 16. Culture of Vibrio Fischeri (prepared with 2216E medium)

|  |  |
| --- | --- |
| Peptone | 5.0 |
| Yeast powder | 1.0 |
| Ferric citrate | 0.1 |
| Sodium Chloride | 19.45 |
| Magnesium Chloride | 5.98 |
| sodium sulfate | 3.24 |
| calcium chloride | 1.8 |
| dicalcium phosphate | 0.55 |
| sodium carbonate | 0.16 |
| potassium bromide | 0.08 |
| strontium chloride | 0.034 |
| boric acid | 0.022 |
| sodium silicate | 0.004 |
| sodium fluoride | 0.0024 |
| ammonium nitrate | 0.0016 |
| disodium hydrogenphosphate | 0.008 |
| pH: 7.6±0.2 | 25℃ |

Weigh 37.4 g of this product, heat and dissolve in 1000 ml of distilled water, 121 ℃ autoclave sterilization for 15 minutes, standby.

Note: The medium has more inorganic salt components and is prone to precipitation after autoclaving, dissolution before sterilization is critical, add water first, add dry powder slowly while stirring, don't let the dry powder form a ball; after sterilization, it should be shaken well, try to let the precipitation disperse, avoid gathering together to affect the experiment.

Inoculate with the Vibrio fischeri and place in aerobic culture at 20-25°C for 40-72 hours.

# 1.Vibrio fischeri culture

The 2216E medium was used to recover Vibrio fischeri lyophilized powder. The medium inoculated with Vibrio fischeri was incubated at 22℃ for 48h, and the luminescence test was carried out using a multifunctional microplate reader after observing the turbidity of the medium and the blue fluorescence in the darkness with the naked eye.

![Gut-sweeper](https://static.igem.wiki/teams/5210/result-1.png)

**Fig 1.****Vibrio fischeri successfully cultured and fluoresced in the laboratory**
A.2216E Culture Media Formulation;
B.Original camera shot luminescent Vibrio fisheri;
C.Multifunctional microplate reader luminescence test.

We successfully cultured Vibrio fischeri in the lab and observed bioluminescence. However, because of the slow growth rate and the time required for luminescence, we did not consider Vibrio fischeri to be suitable as our chassis bacteria, and its luminescence principle inspired us to explore further.

# 2.Envz-OmpR Validating of the Two-component osmotic pressure sensing system Envz-OmpR

Validation experiments were performed with BL21(DE3). LB medium with different NaCl concentrations was prepared and reporter gene expression was observed.

![Gut-sweeper](https://static.igem.wiki/teams/5210/result-2.png)

**Fig 2.****Fluorescence Intensity with Different osmotic pressure LB cocultured in Pompc-EGFP engineered bacteria**
A.plasmid construction;
B.pre-experiment to explore the suitable growth concentration of experimental bacterium BL21;
C. fluorescence intensity of engineered bacteria under different osmotic pressures;
D. Fluorescence microscope to observe the fluorescence intensity of engineered bacteria under different osmotic pressures.

By observing the intensity of reporter gene expression, we demonstrated the sensitivity of the engineered bacteria to high osmotic pressure.

# 3.Validating luminescence efficacy of Lux gene clusters

The Lux gene cluster (LuxCDABE or LuxCDABEGF) was introduced into BL21 (DE3) and expression was induced using IPTG. Bioluminescence was observed by naked eye and photographed. After the obvious luminescence was observed by naked eyes, the fluorescence intensity was quantified using a multifunctional microplate reader.

![Gut-sweeper](https://static.igem.wiki/teams/5210/result-3.png)

**Fig 3.Luminescence results of optimized luminescent gene circuits**
A.Plasmid construction;
B.Multifunctional microplate reader luminescence test;
C.Original camera shot luminescent engineering bacteria;
D.Exposure 15s shot luminescent engineering bacteria.

Lux luminescent gene clusters emit blue fluorescence that is visible to the naked eye, however, it can only be observed in dark environments, and we would like to further enhance the intensity of the luminescence.

# 4.Validating cold-tolerant module protein expression

Plasmids containing proteins encoding SOD and Catalase were introduced into BL21 (DE3) and expression was induced using 0.5 mM and 0 mM IPTG, respectively, and SDS-PAGE was performed to verify protein expression.

![Gut-sweeper](https://static.igem.wiki/teams/5210/result-4.png)

**Fig 4.****Prokaryotic expression of SOD and catalase proteins**
A.Plasmid construction;
B.SDS-PAGE image;
C.SDS-PAGE strip analysis results.

We successfully expressed two proteins, but the amount of expression was not ideal. In the future, we will further explore to optimize the expression and test the engineered bacteria for low temperature resistance.

# 5.Preparation and recovery of lyophilized bacterial powder

BL21 (DE3) was lyophilized using liquid LB medium with 10% sucrose as a protective agent. After one week of lyophilization and preservation, the plates were resuscitated using liquid LB medium, diluted 3,000-fold, coated and cultured in microtiter plates. The coated plates and culture results were visualized.

![Gut-sweeper](https://static.igem.wiki/teams/5210/result-5.png)

**Fig 5.****Results of preparation of lyophilized bacterial powders and resuscitation**
A.Freeze-dried bacterial powder product;
B.Diluted coated plate result after resuscitation;
C.Visualization of coated plate result;
D.Measurement of growth curve after resuscitation.

# 6.Verificating radiation resistance of RecO, RecF

Plasmids containing genes encoding RecO and RecF were introduced into BL21 (DE3), diluted coated plates (agar plates containing IPTG) were irradiated with different doses of UV light, and surviving colonies were counted after 3 days of incubation. Protein expression was induced with 1 mM IPTG and SDS-PAGE was performed to verify protein expression.

![Gut-sweeper](https://static.igem.wiki/teams/5210/result-6.png)

**Fig 6.****Results of prokaryotic expression of RecO and RecF genes and validation of radiation resistance**
A.Plasmid construction;
B.Changes in the survival of bacteria after UV irradiation at different doses;
C.SDS-PAGE image.

We successfully expressed RecO and RecF proteins in BL21(DE3) and verified the survivability of the engineered bacteria with UV irradiation.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

### Results

Scroll To Explore

![]()

# Overview

After a year of concerted efforts and unremitting efforts, the genetic route we designed was basically
successful. The growth and luminescence of the algae-fungus mixture system were modeled and verified. In
addition, we explored the future application scenarios of the product and developed improvement
strategies to put our project into use as soon as possible.

* [**Experiment**
  Experiment results](#r1)
* [**Model**
  Model Results](#r2)
* [**Product**
  Product design and future plans](#r3)
  + [Product](#r3)
  + [Application Space](#Space)
  + [Application Earth](#Earth)
* ![糖豆人](https://static.igem.wiki/teams/5210/logo-astronaut-2.png)

  ![](https://static.igem.wiki/teams/5210/logo/logo-5.png)

# Experiment

## 1.Vibrio fischeri culture

The 2216E medium was used to recover Vibrio fischeri lyophilized powder. The medium
inoculated with Vibrio fischeri was incubated at 22℃ for 48h, and the luminescence test
was carried out using a multifunctional microplate reader after observing the turbidity
of the medium and the blue fluorescence in the darkness with the naked eye.

![Gut-sweeper](https://static.igem.wiki/teams/5210/result-1.png)

**Figure. 1 |** **Vibrio fischeri successfully cultured
and fluoresced in the laboratory**

A. 2216E Culture Media Formulation;
B. Original camera shot luminescent Vibrio fisheri;
C. Multifunctional microplate reader luminescence test.

We successfully cultured Vibrio fischeri in the lab and observed bioluminescence.
However, because of the slow growth rate and the time required for luminescence, we did
not consider Vibrio fischeri to be suitable as our chassis bacteria, and its
luminescence principle inspired us to explore further.

## 2.Envz-OmpR Validating of the Two-component osmotic pressure sensing system Envz-OmpR

Validation experiments were performed with BL21(DE3). LB medium with different NaCl
concentrations was prepared and reporter gene expression was observed.

![](https://static.igem.wiki/teams/5210/result-2.png)

**Figure. 1 |**
 **Fluorescence Intensity with Different osmotic pressure LB cocultured in Pompc-EGFP engineered bacteria**

A. plasmid construction;
B. pre-experiment to explore the suitable growth concentration of experimental bacterium BL21;
C. fluorescence intensity of engineered bacteria under different osmotic pressures;
D. Fluorescence microscope to observe the fluorescence intensity of engineered bacteria under different osmotic pressures.

## 3.Validating luminescence efficacy of Lux gene clusters

The Lux gene cluster (LuxCDABE or LuxCDABEGF) was introduced into BL21 (DE3) and
expression was induced using IPTG. Bioluminescence was observed by naked eye and
photographed. After the obvious luminescence was observed by naked eyes, the
fluorescence intensity was quantified using a multifunctional microplate reader.

![](https://static.igem.wiki/teams/5210/result-3.png)

**Figure. 3 |**
**Luminescence results of optimized luminescent gene circuits**

A. Plasmid construction;
B. Multifunctional microplate reader luminescence test;
C. Original camera shot luminescent engineering bacteria;
D. Exposure 15s shot luminescent engineering bacteria.

Lux luminescent gene clusters emit blue fluorescence that is visible to the naked eye, however, it can only be observed in dark environments, and we would like to further enhance the intensity of the luminescence.

## 4.Validating cold-tolerant module protein expression

Plasmids containing proteins encoding SOD and Catalase were introduced into BL21 (DE3) and expression was induced using 0.5 mM and 0 mM IPTG, respectively, and SDS-PAGE was performed to verify protein expression.

![](https://static.igem.wiki/teams/5210/result-4.png)

**Figure. 4 |**
**Prokaryotic expression of SOD and catalase proteins**

A. Plasmid construction;
B. SDS-PAGE image;
C. SDS-PAGE strip analysis results.

We successfully expressed two proteins, but the amount of expression was not ideal. In the future, we will further explore to optimize the expression and test the engineered bacteria for low temperature resistance.

## 5.Preparation and recovery of lyophilized bacterial powder:

BL21 (DE3) was lyophilized using liquid LB medium with 10% sucrose as a protective agent. After one week of lyophilization and preservation, the plates were resuscitated using liquid LB medium, diluted 3,000-fold, coated and cultured in microtiter plates. The coated plates and culture results were visualized.

![](https://static.igem.wiki/teams/5210/result-5.png)

**Figure. 5 |**
**Results of preparation of lyophilized bacterial powders and resuscitation**

A. Freeze-dried bacterial powder product;
B. Diluted coated plate result after resuscitation;
C. Visualization of coated plate result;
D. Measurement of growth curve after resuscitation.

## 6.Verificating radiation resistance of RecO, RecF

Plasmids containing genes encoding RecO and RecF were introduced into BL21 (DE3), diluted coated plates (agar plates containing IPTG) were irradiated with different doses of UV light, and surviving colonies were counted after 3 days of incubation. Protein expression was induced with 1 mM IPTG and SDS-PAGE was performed to verify protein expression.

![](https://static.igem.wiki/teams/5210/result-6.png)

**Figure. 6 |**
**Results of prokaryotic expression of RecO and RecF genes and validation of radiation resistance**

A. Plasmid construction;
B. Changes in the survival of bacteria after UV irradiation at different doses;
C. SDS-PAGE image.

We successfully expressed RecO and RecF proteins in BL21(DE3) and verified the survivability of the engineered bacteria with UV irradiation.

# Model

| Model | Method or Equation | Results |
| --- | --- | --- |
| Disaster Prediction Model (DPM) | | |
| ARIMA | ![](https://static.igem.wiki/teams/5210/result-7.png) | July is the time of high incidence of accidents every year. It was predicted that the next accidents would occur at around 0:00 at night. |
| Random Forest Regression Algorithm | Utilizing the relationship between historical time differences and geographical coordinates to separately predict the latitude and longitude of future accident locations | The distribution of latitudes and longitudes is relatively dispersed. Fitting effect: Latitude Model R²: 0.791, Longitude Model R²: 0.702. |
| Luminous Life-Saving Model (LSM) | | |
| Population growth model | Synechococcus: ![](https://static.igem.wiki/teams/5210/result-8.png) Vibrio natriegens: ![](https://static.igem.wiki/teams/5210/result-9.png) | Synechococcus enters the exponential growth phase after 10 hours and reaches the plateau phase around 40 hours; V. natriegens grows slowly in the first 30 hours, and the growth rate accelerates significantly after 40 hours. |
| Luminous line simulation | ![](https://static.igem.wiki/teams/5210/result-10.png) | The illuminance changes slightly in the first 20 hours, and then increases rapidly over time, reaching a peak of 1567 lx at 48.87h. As the number of V. natriegens and Synechococcus stabilizes, the illuminance remains at about 1000 lx. It is estimated that the maximum coverage radius can reach about 90 meters on the sea. |
| VeloVI model | It re-infers the RNA velocity through a model that shares information among all cells and genes, while learning kinetic parameters and latent time | Output the kinetic parameters such as transcription rate of the corresponding gene. |
| Sensitivity analysis | On the basis of the originally set luminous efficiency coefficient value (η) of 30%, it is adjusted up and down by 20% to simulate the effect of external regulatory factors (such as pH, temperature, etc.) | The model is robust and the overall trend remains unchanged. When the value of η is increased, the peak value of I is significantly improved, and the luminous effect decreases after 50 hours; when the value of η is decreased, the illumination intensity fluctuates less after reaching the peak value, which is superior in long-term rescue. |
| Genetic Algorithm | It consists of three basic operations: selection, crossover, and mutation. By simulating natural selection and genetic mechanisms, it searches for the optimal solution in the search space | The optimal illumination intensity (I) is 4582.49 lx, which is an increase of 192.4% compared to the initial state. This provides support for exploring the luminous potential of the system. |
| Space Feasibility Analysis (SFA) | | |
| Rank-Sum Ratio (RSR) Comprehensive Evaluation Method | Collect and analyze data of various planets in the solar system from multiple angles. Obtain dimensionless statistical quantity RSR through rank transformation, and sort or grade the evaluation objects directly according to the RSR value, so as to make a comprehensive evaluation of the evaluation objects | The Bio-Beacon Rescue Locator is most suitable for application on Mars due to its small difference from the Earth's environment in terms of rotation period, water resources and other indicators. |
| Molecular Docking Test of Repressor Protein | | |
| Molecular docking technique | The structure files of sucrose, corresponding protein and DNA were obtained by the tool, and 240ps molecular dynamics simulation was performed after the global docking of proteins, and the binding of proteins and DNA was analyzed before and after the simulation. | The affinity between repressor protein and DNA did not change significantly before and after sucrose treatment. Further analysis of the results showed that changes in binding mode enabled DNA exposure to be transcribed, demonstrating that repression could be inhibited after sucrose treatment. |

# Product design and future plans

Based on the algal bacterial system, we have designed fluorescent positioning life jackets, parachutes and space suits to adapt to different application scenarios.

①Fluorescent positioning life jacket: In case of shipwreck or emergency, the fluorescent positioning life jacket can quickly respond to environmental changes, and the built-in freeze-dried bacterial powder package emits blue fluorescence when activated by water, significantly improving the rescue positioning efficiency and earning valuable survival time for those in distress.

②Fluorescent positioning parachute: When the aircraft or helicopter is forced to land on the sea for some reason, the biological coating integrated on the surface of the fluorescent positioning parachute can be activated and emit blue fluorescence after encountering water, providing accurate positioning for search and rescue operations.

③Ecological rescue spacesuit: When operating on the surface of a planet or performing a mission in the event of an accident, the ecological rescue spacesuit can emit bright fluorescent signals for search and rescue, while its miniature ecosystem can continue to provide astronauts with oxygen and nutrients needed for life, greatly extending the survival time of astronauts, adding a new dimension of safety to space exploration.

![](https://static.igem.wiki/teams/5210/result-product-1.png)

In fact, LuminAid is used in a wide range of applications. Our initial aim was to apply this technology to spacesuits, so as to make up for some of the shortcomings of existing bionic lighting technology, reduce the energy dependence on the earth, and achieve in-situ and sustainable use of energy; It can also be equipped to the outside of the rescue chamber as a kind of identification mark. At the same time, the new technologies applied in space exploration can often feed back to the earth, providing new focus for the development of earth science and technology. We focused on tapping into the huge potential of LuminAid in terraforming and designed it to be equipped with life jackets, parachutes, and various rescue scenarios.

## 1. Space

### (1)Spacesuit

Deep space exploration missions, planetary surface surveys, long-term spacewalks: In deep space areas far away from Earth and communication signals may be interrupted, if astronauts encounter accidental loss of contact, LuminAid equipped on the spacesuit will become a rescue beacon for rescuers, providing emergency positioning functions.

![Gut-sweeper](https://static.igem.wiki/teams/5210/product-figure1-tihuan.jpeg)

**Figure. 1 |** **Equipped with a LuminAid-equipped spacesuit**

### (2)Space capsule

Capsule Identification and Positioning: LuminAid can emit a large intensity of fluorescence without external energy supply, which can be used for the identification and positioning of the space capsule, which can save other energy.

![Gut-sweeper](https://static.igem.wiki/teams/5210/result-12.png)

**Figure. 2 |** **LuminAid for extracapsule equipment**

## 2. Earth

### (1)Life jacket

Marine navigation and operations, water sports and leisure, and shipwreck rescue: For fishermen, seafarers and offshore oil exploration workers and other personnel who have been working at sea for a long time, surfing, windsurfing, kayaking and other water sports enthusiasts, and victims of shipwrecks such as ship sinking and people falling overboard, LuminAid equipped with life jackets can automatically emit fluorescent signals in an emergency to help search and rescue teams quickly locate them.

![Gut-sweeper](https://static.igem.wiki/teams/5210/result-13.png)

**Figure. 3 |** **Life jackets equipped with LuminAid**

### (2)Parachute

Maritime Disaster Rescue: When an airplane or helicopter is forced to land on the sea surface for some reason, passengers and crew can use this parachute to escape urgently. The fluorescent effect of the parachute surface when it encounters water can quickly attract the attention of search and rescue personnel and shorten the rescue time.

![Gut-sweeper](https://static.igem.wiki/teams/5210/product-figure4-tihuan.jpeg)

**Figure. 4 |**
**Equipped with a parachute of LuminAid**

### (3)Others

In addition to this, our Luminaid algal system can be used in a number of scenarios: Polar research, mineral mining, deep-sea exploration, marine waste marking...

![Gut-sweeper](https://static.igem.wiki/teams/5210/result-15.png)

**Figure. 5 |**
**Luminaid is used in the mining of mineral sources**

![Gut-sweeper](https://static.igem.wiki/teams/5210/result-16.png)

**Figure. 6 |**
**Use Luminaid in deep-sea expeditions**

{% endblock %}