iGEM 2023 — Team fzpt-china

{% extends "layout.html" %}
{% 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 %}
{% block page\_content %}

## What should this page contain?

---

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block page\_content %}

# Engineering

↓

---

[Overview](#Overview)

[Design](#Design)
[Design Cycle 1](#Cycle1)
[Design Cycle 2](#Cycle2)
[Design Cycle 3](#Cycle3)
[Design Cycle 4](#Cycle4)
[Design Cycle 5](#Cycle5)

[Summary](#Summary)

#### Overview

##### （Why-How-What-Which）

##### Why did we choose this project？

#####

Firstly, we were inspired by the predecessors who utilized the microalgae-bacteria
interaction system for sewage treatment and aim to exploit the ecological interplay between bacteria and
microalgae to facilitate ecological restoration. The two organisms can mutually influence each other's growth,
metabolism, and function through the exchange of matter and information. However, their interaction can vary
from mutualistic or symbiotic to antagonistic, parasitic, or competitive depending on their respective needs
and environmental conditions. Based on this characteristic, we simulate a symbiotic relationship between
*Phaeodactylum tricornutum* and *Qipengyuania aquimaris* in nature through co-cultivation. Our
goal is to
determine an optimal proportion that facilitates the growth of marine Vibrio on *P. tricornutum* while
examining
its effects on nutrient metabolism, lipid accumulation, and other related aspects.

##### How can  we improve the current project？

#####

In order to enhance the analysis of the interaction between *P. tricornutum*
and Q.
aquimaris in co-culture, we intend to further investigate their interaction using co-culture and separation
culture methods based on our team's previous exploration of microbial immobilization schemes. This will enable
us to identify more precise co-culture strategies. Additionally, due to material constraints, the initial
microbial immobilization solution relied on manual embedding and preparation methods, resulting in suboptimal
efficiency. Consequently, we aim to develop supplementary hardware equipment aligned with microbial
immobilization scheme this year.

##### What genetic manipulation  is planned for the project？

#####

Drawing inspiration from the 2021 CHINA-FAFU project, which focused on exogenously
adding L-ascorbic acid and employing synthetic biology to overexpress genes related to the synthetic pathway
in order to enhance antioxidant capacity of microalgae and evaluate its impact on photosynthesis and lipid
accumulation, our study aims to investigate the effects of another specialized antioxidant substance that
promotes endogenous synthesis of fucoidan in microalgae. Specifically, we aim to explore its influence on
nitrogen-phosphorus utilization, response to photosynthetic stress, as well as lipid accumulation in
microalgae.

##### Which is expect from this  genetic manipulation？

#####

    Fucoidan, an isomer composed of fucoturonic acid and fucosamine, exhibits remarkable
biological properties including potent antioxidant activity, immunomodulatory effects, and hypoglycemic.
Concurrently, fucosidase is an enzyme capable of hydrolyzing fucosidic bonds that connect fucoturonic acid and
fucosamine. This enzyme can be found in certain marine micro-organisms and plays a role in the degradation or
modification of fucoidan. Therefore, our project aims to clone and overexpress the fucosidase gene from P.
tricornutum by using synthetic biology techniques. This approach may lead to the development of transgenic
microalgae strains with enhanced antioxidant capacity, nitrogen-phosphorus nutrient absorption efficiency,
lipid accumulation ability, among other desirable traits. Ultimately, it offers novel options for
bioremediation through bacteria-microalgae interactions by introducing new algal species and programs into
practice.

![](https://static.igem.wiki/teams/4688/wiki/engineer2.png)

Figure 1 Technology Roadmap

In our project, we rigorously adhere to the iterative
“Design-Build-Test-Learn
cycle” to optimize our
biological systems and achieve the desired functionality and performance through continuous iterations of
design, construction, testing, and learning.

![](https://static.igem.wiki/teams/4688/wiki/fgtwo.png)

#### Design

##### Design Cycle 1： Optimizing the ratio of bacteria-microalgae interaction

**Design:**

The bacteria-microalgae ratio refers to the proportion of biomass or cell number between bacteria and
microalgae in the interaction system. It is a crucial factor that affects the stability and efficiency of the
symbiotic relationship. Different ratios can lead to varying material exchanges and information transmissions,
thereby influencing growth, metabolism, and function of this interaction system.

Generally speaking, a lower bacteria-microalgae ratio benefits the growth of microalgae, as bacteria can
provide regenerative nutrients and growth stimulants, while also suppressing the natural enemies and pathogens
of microalgae. Conversely, a higher ratio favors the growth of bacteria as microalgae provide light energy
sources, as well as increase the pH and dissolved oxygen content of the water.

However, it should be noted that this rule is not universally applicable in all cases. Several studies have
demonstrated that different types or states of bacteria or microalgae exhibit varying adaptability and
sensitivity to the bacteria-microalgae ratio, sometimes yielding contradictory outcomes. For instance, certain
bacteria possessing nitrogen fixation ability or antibiotic production capabilities can stimulate microalgae
growth at higher bacteria-algae ratios. Similarly, heat or salt-tant microalgae producing protective
substances may enhance bacterial proliferation at lower bacteria-microalgae ratios.

**Build:**

Consequently, determining the optimal bacteria-microalgae ratio necessitates considering multiple factors,
such as the type, characteristics, state, and environmental conditions of both bacteria and microalgae. In our
project, we aimed to conduct experiments and modeling by employing density ratios between microalgae and
bacteria.

![](https://static.igem.wiki/teams/4688/wiki/egntwo.png)

Figure 2 Experimental schematic diagram of the
interaction ratio between
different bacteria and microalgae

**Test:**

We designed a gradient density ratio experiment based the characteristics of experimental materials. P.
tricornutum and *Q. aquimaris* were cultured in ratios of 1:1, 1:3, 1:5, 1:7, and 1:9 respectively,
while
conventional culture conditions were employed for microalgae as a control group. In this study, we determined
an optimal symbiotic ratio by considering factors such as biomass, lipid accumulation, and photosynthetic
efficiency.

![](https://static.igem.wiki/teams/4688/wiki/egnthree.png)

Figure 3 Effect of different
microalgae-bacteria interaction ratios on lipid
accumulation in microalgae

**Learn:**

During Design Cycle 1, we observed that diverse bacteria-microalgae interactions can potentially impede
nitrogen-phosphorus removal in the water while concurrently facilitating biomass accumulation in P.
tricornutum. However, these interactions exhibit a favorable impact on lipid accumulation and overall biomass
growth in microalgae.

![](https://static.igem.wiki/teams/4688/wiki/fgone.png)

##### Design Cycle 2： Optimizing embedding conditions for bacteria-microalgae interaction system and developing supporting hardware

**Design:**

In the bacteria-microalgae interaction system, mutual interactions between *P. tricornutum* and *Q.
aquimaris*
can
occur through material exchange, information transmission, and structure formation. To further investigate
this system, we employed a commonly approach involves co-culture and separation culture for comparative
analysis.

**Build:**

We implemented microbial immobilization embedding schemes based on previous research findings to investigate
the forms of interaction between *P. tricornutum* and *Q. aquimaris*. Furthermore, in order to
minimize
potential
confounding factors associated with microbial immobilization embedding schemes, we designed relevant
hardware
equipment and auxiliary devices.

**Test:**

In terms of designing the experimental group, we established a total of five treatment groups to investigate
interactive effects under different cultivation conditions. Microalgae and bacteria were co-cultivated in an
optimal ratio (AB). Microalgae were individually immobilized and co-cultivated with bacteria(AO）, while
bacteria were individually immobilized and co-cultivated with microalgae(BO). Additionally, bacteria and
microalgae were immobilized separately(ABO) or together(ABS) , and co-cultivated subsequently.

![](https://static.igem.wiki/teams/4688/wiki/egnfour.png)

Figure 4 Experimental schematic diagram
comparing different immobilization
strategies for the interaction between bacteria and microalgae

The consumption rate of total nitrogen in the culture medium along with the accumulation of lipids and
pigments in microalgae were utilized as evaluation indicators. Regrettably, during the initial design phase,
we overlooked the challenges associated with sampling and detection in the immobilization system, which
hindered some anticipated evaluation indicators from proceeding normally, potentially leading to an
incomplete
assessment process.

![](https://static.igem.wiki/teams/4688/wiki/egnfive.png)

Figure 5 Experimental Phenotype Diagrams

Fortunately, our hardware device version 1.0 designed for microbial embedment proved instrumental in this
evaluation experiment. The utilization of this device for producing microbial immobilized beads resulted in
more uniform spheres prepared at a faster pace compared to manual embedding, significantly reducing time
costs.

![](https://static.igem.wiki/teams/4688/wiki/egnsix.png)

Figure 6 Effects of different immobilization
interaction modes on lipid
accumulation in microalgae

![](https://static.igem.wiki/teams/4688/wiki/egnseven.png)
![](https://static.igem.wiki/teams/4688/wiki/hardware/f33.gif)

Figure 7 Microbial Embedding Device v1.0

**Learn:**

Detecting lipids and pigments of immobilized interaction system poses a challenging task. We acknowledge
that
a small part of cells rupture caused by sodium citrate during dissociation of immobilized balls is
temporarily
inevitable.

Surprisingly, our findings reveal that separate immobilization culture of bacteria（BO） exhibit a significant
reduction in microalgae lipid accumulation but a notable increase in the chloroplast volume and pigment
accumulation compared to control group.

Furthermore, we also realize that there are some issues with the current microbial embedding device:
potential
operational risks associated with high voltage (110v), long delivery pipelines (resulting in certain wastage
of embedding materials), and relatively complex operational logic requirements. **Based on these
feedback, our
experimental team intends to implement iterative updates addressing these concerns.**

![](https://static.igem.wiki/teams/4688/wiki/fgtwo.png)

##### Design Cycle 3：Clone new components and verify their successful working in microalgae cells.

**Design:**

The Design Cycle 1 revealed that different species exhibit varying efficiencies in nutrient absorption,
such as nitrogen and phosphorus. Consequently, the utilization of bacteria-microalgae interaction for
water remediation may not yield the desired outcomes.

In order to fully leverage the beneficial characteristics of bacteria-microalgae interaction, while also
addressing the rapid absorption and utilization requirements for elements like nitrogen and phosphorus in
aquatic environments. To achieve this, we intend to employ synthetic biology techniques starting from
microalgae for modification.

**Build:**

Base on the previous research on enhancing the capacity and lipid accumulation of microalgae, we have cloned
and constructed and overexpression systems for three isozymes of fucosidase (BBa\_K4688002 to BBa\_K4688004) in
the genome of *P. tricornutum*，which were subsequently labeled with fluorescence for convenient
localization.

![](https://static.igem.wiki/teams/4688/wiki/egneight.png)

Figure 8 Schematic diagram of engineering strain construction
and transformation process

**Test:**

By designing specific primers, we successfully amplified and cloned BBa\_K4688002 to BB\_K4688004. Subsequently,
utilizing the infusion method, we ligated the obtained fragments into the expression vector provided by the
2022 CHINA-FAFU team. Finally, microalgae strains were validated at the DNA, RNA, and Protein levels.

![](https://static.igem.wiki/teams/4688/wiki/egnnine.png)

Figure 9 Cloning of the target gene

![](https://static.igem.wiki/teams/4688/wiki/egnten.png)

Figure 10 Colony PCR
validation of the overexpression system
constructed in microalgae

（*PtFUCO1* (A),*PtFUCO2*(B), *PtFUCO3* (C)overexpression
vectors）

**Learn:**

In the construction of engineered microalgae strains, the electroporation introduces some instability in the
transforming efficiency, which will be further optimized to enhance transformation. Fortunately, we have
successfully obtained an overexpressed engineered microalgae strain for one of the fucosidases.

![](https://static.igem.wiki/teams/4688/wiki/fgthree.png)

##### Design Cycle 4：Evaluation of engineered microalgae strains and the application of new embedding hardware.

**Design:**

Based on the engineered microalgae strains obtained in Design Cycle 3 and relevant conclusions combined with
Design Cycles 1-2, we evaluated the physiological and biochemical indicators of FUCO3 microalgae strain.

**Build:**

We conducted experiments to evaluate the environmental remediation performance of individual engineered
microalgae strains and bacteria-microalgae interaction system comprising these engineered microalgae strains
in conventional culture media or artificially simulated wastewater. Furthermore, we employed microbial
embedding device v2.0 to facilitate the execution of these experiments.

![](https://static.igem.wiki/teams/4688/wiki/egnele.png)

Figure 11 Experimental schematic diagram for testing
themediation performance of engineered microalgae

![](https://static.igem.wiki/teams/4688/wiki/egntwl.png)

Figure 12 Microbial Embedding Device v2.0

**Test:**

After testing, we observed significant enhancements in the nitrogen removal efficiency, lipid accumulation and
photosynthetic efficiency of the engineered microalgae strains when cultivated individually or in the
bacteria-microalgae interaction system. Furthermore, the improved microbial embedding device 2.0 exhibited
enhanced safety (12v), more streamlined pipeline design, and more user-friendly operational logic during
experimentation, thereby providing favorable hardware support for microbial immobilization.

**Learn:**

Due to the exceptional characteristics of the engineered strain of microalgae, we aim to further elucidate the
molecular mechanisms underlying algal lipid accumulation, nutrient element utilization (specifically nitrogen
and phosphorus), as well as fucoxanthin accumulation mediated by fucosidase. Moreover, we intend to explore
its potential commercial value.

![](https://static.igem.wiki/teams/4688/wiki/fgfour.png)

##### Design Cycle 5：Futher molecular mechanism analysis of genetically modified microalgae strains

**Design:**

Given the outstanding performance of BBa\_k4112013 and BBa\_k4112014 in microalgae bioremediation utilizing
which designed by our former CHINA-FAFU team, we aim to further explore the potential bioremediation
characteristics achievable by integrating our previous research with the ongoing development of engineered
microalgae and bacteria-microalgae interaction system.

**Build:**

We intend to conduct comprehensive validation of the transfer of BBa\_k4112013 and BBa\_k4112014 vectors into
appropriate engineered microalgae strains during Design Cycle 3. Furthermore, we enhance the bioremediation
strategies by integrating these engineered microalgae strains into a bacteria-microalgae interaction system.

**Test:**

We have completed the transformation of engineering microalgae strains at present, but due to the
time-consuming growth cycle and identification process of microalgae, we have not yet achieved positive
outcomes. Nevertheless, our research in this field will persist.

#### Summary

In this prestigious competition, our team meticulously selected a topic aligned with our interests and
skillfully components along with standard molecular to construct and rigorously test a sophisticated
biological system.

First and foremost, we would like to express my heartfelt gratitude to the organizing committee and the
competition platform for providing us with this exceptional opportunity to showcase our innovative
achievements and research capabilities.Throughout the entire process, we diligently adhere to the iterative
“Design-Build-Learn cycle” by continuously testing, and learning. This approach guided us inrefiningour
project through a systematic and iterative refinement process.

Secondly, we extend our sincere appreciation to our esteemed professors and scholars who have been
instrumental in guiding us on this journey. Their invaluable advice and feedback have enabled us to achieve
breakthroughs both technically and theoretically. Their expertise and extensive experience have profoundly
influenced our learning and growth.

Lastly, we want to convey our deep appreciation for our remarkable team members who have proven themselves as
outstanding collaborators. We worked together cohesively during the competition, supporting each other's
progress every step of the way. This competition not only enhanced our research abilities but also improved
our teamwork skills and communication prowess. We gained a wealth of knowledge and experience while forging
many new friendships along this incredible journey.

Overall, we attribute our accomplishments in the iGEM competition to both its inherent excellence as well as
the exceptional support rendered by our guiding professors and scholars who enabled us to effectively navigate
through every stage of the Design Cycle involved in this ambitious endeavor.

**If you want to learn more about other aspects of our engineering journey have a look at our [Hardware](https://2023.igem.wiki/fzpt-china/hardware)
and [Results](https://2023.igem.wiki/fzpt-china/results)
page!**

{% endblock %}

FAFU

# Experiments

## General

Primers

The nucleotide sequence of the target gene containing the
upstream and downstream fragments was selected from the genome
sequence of *Phaeodactylum tricornutum*, and then the upstream
and
downstream homologous arm primers (upstream and downstream
homologous arm forward and reverse primers) were designed by
using PrimerPremier5.0 at about 500bp of the upstream and
downstream fragments. At the same time, the forward and reverse
primer pairs of the target gene were designed, and the primers
were designed at the left and right of the front end of the
100bp of the upstream homologous arm, respectively. And the
reverse primers of the downstream homologous arm were designed
at the left and right of the back end of 100bp. The primer
sequence is shown in the attachment.

Agar plate

#### LB- Agar plate

1. Place 10g Tnypxone, 5g Yeast extract, 10g NaCl, 15g Ager, in
   1L of up-water.
2. Autoclave at 121℃ for 20min.
3. Add the required antibiotics after cooling at room
   temperature, mix and pour into disposable sterile Petri
   dishes and store at 4℃.

#### F/2- Agar plate

1. 75 mg NaNO3, 5 mg
   NaH2PO4-H2O, 4.36 mg
   Na2EDTA-2H2O, 3.16 mg
   FeCl3-6H2O, 0.01 mg
   CuSO4-5H2O, 0.023 mg
   ZnSO4-7H2O, 0.012
   mg COCl2-6H2O, 0.018 mg
   MnCl2- 4H2O, 0.07mg
   NaMoO4-2H2O,
   0.1×10-3mg vitamin B1, 0.5×10-6mg
   biotin (vit. H),
   0.5×10-6mg vitamin B12, 15g Ager placed in 1L of
   seawater.
2. Autoclave at 121℃ for 20min.
3. Add the required antibiotics after cooling at room
   temperature, mix and pour into disposable sterile Petri
   dishes and store at 4℃.

Liquid medium

#### LB- medium plate

1. Place 10g Tnypxone, 5g Yeast extract, 10g NaCl, in 1L of
   dd-water.
2. Autoclave at 121℃ for 20min.
3. Add the required antibiotics after cooling at room
   temperature, mix and pour into disposable sterile Petri
   dishes and store at 4℃.

#### F/2- medium plate

1. 75 mg NaNO3, 5 mg
   NaH2PO4-H2O, 4.36 mg
   Na2EDTA-2H2O, 3.16 mg
   FeCl3-6H2O, 0.01 mg
   CuSO4-5H2O, 0.023 mg
   ZnSO4-7H2O, 0.012
   mg COCl2-6H2O, 0.018 mg
   MnCl2- 4H2O, 0.07mg
   NaMoO4-2H2O,
   0.1×10-3mg vitamin B1, 0.5×10-6mg
   biotin (vit. H),
   0.5×10-6mg vitamin B12,placed in 1L of
   seawater.
2. Autoclave at 121℃ for 20min.
3. Add the required antibiotics after cooling at room
   temperature, mix and pour into disposable sterile Petri
   dishes and store at 4℃.

Statistical analysis

Statistical analysis was performed with
the
Student's
paired
t-test. All assays were performed independently for a minimum of
three biological replications. Data are represented as mean ±
SD.

## Microalgae&Bacteria

Algal species and culture

#### Algal species

*Phaeodactylum tricornutum*（*P. tricornutum*） is
provided by the
CCMP algae repository in the United States, under the
species label CCMP2561.

#### Algal culture

The marine diatoms *P. tricornutum* were grown in batches in
triangular conical flasks containing f/2-Si medium and
incubated in an artificial climate chamber. The parameters
of the artificial climate chamber were: temperature 21 ±
1°C, photoperiod 12 h/12 h (L/D), and light intensity 150
μmol photon m-2 s-1.

The 100 mL of algal solution was incubated in 250 mL
triangular conical flasks and the mouths of the flasks were
sealed with a sealing film containing 0.22 μm filter
membrane to isolate airborne bacteria and other materials.
The algal solution was manually shaken three times a day
during the incubation period to slow down the aggregation of
cells settling in the triangular conical flask.

Bacteria species and culture

#### Bacteria species

*Qipengyuania aquimaris* is provided by MCCC (Marine Culture
Collection of China), under the species label MCCC 1A14896.

#### Bacteria culture

The marine bacteria *Qipengyuania aquimaris* were grown in
triangular conical flasks containing 2216e culture medium,
incubated in an artificial climate chamber. The parameters
of the artificial climate chamber were: temperature 21 ±
1°C, photoperiod 12 h/12 h (L/D), and light intensity 150
μmol photon m-2 s-1.

The 100 mL of bacteria solution was incubated in 250 mL
triangular conical flasks and the mouths of the flasks were
sealed with a sealing film containing 0.22 μm filter
membrane to isolate airborne bacteria and other materials.
The bacteria solution was manually shaken three times a day
during the incubation period to slow down the aggregation of
cells settling in the triangular conical flask.

Algae and bacteria symbiosis

#### Interactions between microalgae and bacteria

For the ratio of microalgae to bacterial cell density in
culture, we set up six experimental groups: R1(microalgae:
bacteria = 1:0, microalgae only) , R2(microalgae: Bacteria =
1:1) , R3(microalgae: Bacteria = 1:3) , R4(microalgae:
bacteria = 1:5) , R5(microalgae: bacteria = 1:7) ,
R6(microalgae: bacteria = 0:1, bacteria only) .

Microalgae & Bacteria cell count and growth curve
determination

#### Microalgae & Bacteria cell count

The cell density of *P. tricornutum* was observed and counted
by hemocytometer plate and light microscope analysis.

The density formula was CD = (N/80) × 400 × 10^4, where N is
the total number of cells in 80 small compartments of the
hemocytometer plate.

The optical density of *Phaeodactylum tricornutum* was
measured at the wavelength of 680 nm, and the optical
density of bacteria was measured at 600 nm.

#### Microalgae & Bacteria growth curve determination

At least 400 cells were counted by light microscopy with a
hemocytometer plate and cell growth curves were plotted for
each treatment group at regular intervals daily during the
*Phaeodactylum tricornutum* triangle culture cycle.

Microalgae & Bacteria cell enrichment

Enrichment of 500 ml of algal or bacteria solution was
performed using a sterile 50 ml centrifuge tube at 4400 rpm,
4°C for 10 min. The supernatant was discarded, the
precipitate was rinsed with RO water, the supernatant was
discarded by centrifugation, and the procedure was repeated
three times. The collected algal precipitate was transferred
to 1.5 ml EP tubes and snap-frozen in liquid nitrogen,
followed by freeze-drying for 12 h or longer until all water
evaporated. Finally, after snap-freezing in liquid nitrogen,
the algal precipitate was immediately stored at -80°C.

Microalgae & Bacteria grinding

Liquid nitrogen is poured into the mortar to pre-cool the
mortar and mortar rod. The enriched algal or bacteria masses
were then poured into the mortar and quickly ground to a
powder, while liquid nitrogen was continuously added to
ensure that the mortar and sample were kept at a low
temperature. Finally, the powder was poured into 1.5 ml
RNAse free EP tubes and placed back into liquid nitrogen for
freezing. The powder was freeze-dried in a freeze-dryer
until all the water was evaporated, then frozen in liquid
nitrogen and stored at -80°C.

Determination of nitrogen and phosphorus content

Reagents:

1. Digestant solution: 20g of potassium persulfate
   (K2S2O8
   2times recrystallized) and 3g of sodium hydroxide (NaOH)
   dissolved in water and diluted to 1000ml.
2. Nitrogen standard storage solution: weigh 0.7218g of
   potassium nitrate (KNO3) dissolved in water
   and diluted
   to 1000ml. This solution is 100ug/ml of nitrate nitrogen
   (NO3-N).
3. Phosphorus standard stock solution: weigh 0.4394g of
   potassium dihydrogen phosphate
   (KH2PO4) baked at 45℃ for
   4-8 hours, dissolved in water and add 1ml (1 + 1) H₂SO₄
   solution and diluted to 1000ml. This solution is
   100ug/ml of phosphorus stock solution.
4. Molybdenum-antimony storage solution of N sulfuric acid:
   Take 180.6 ml of concentrated sulfuric acid
   (H2SO4) and
   add it slowly to 400 ml of water with constant stirring
   and cool. Also weigh ammonium molybdate
   [(NH4)2MoO4-4H2O] 20g
   Dissolve in 300 ml of water at
   approx. 60°C, stir constantly, add 100 ml of 0.5%
   antimony potassium tartrate solution, cool and dilute to
   1000 ml with distilled water, shake well and store in
   brown reagent bottles.
5. Molybdenum-antimony anti-mixed color developer: In 100
   ml of molybdenum-antimony storage solution, add 1.5 g of
   L-ascorbic acid.

    This reagent is effective 24 hours,
should be prepared before use.

Steps:

1. Add a reagent to the nitrogen-phosphorus glass tube.
2. Sample digestion: glass tube cover and tighten the lid,
   wrapped with a sealing film, into the sterilizer 121 ℃,
   digestion for 30 min; digestion is complete, timely
   shaking of nitrogen-phosphorus glass tube in the liquid
   to avoid precipitation undissolved, remove the sealing
   film, and wait for the liquid to cool to room
   temperature.
3. Determination by spectrophotometry: Use Nanodrop, select
   the ultraviolet spectrophotometry program (UV-Vis),
   uncheck the Baseline correction when measuring, use the
   wavelengths of 220 nm and 276 nm for nitrogen
   measurement, and use the wavelength of 700 nm for
   phosphorus measurement, and draw up 2.5 μL of the sample
   each time to carry out the above measurements, and then
   draw a standard curve according to the measured data.
   The standard curve was plotted based on the measured
   data.

Extraction and determination of Fucoxanthin from microalgae

#### Fucoxanthin standard curve

    Take 11 test tubes, number them, add
various solutions according to Table 1, and then measure the
absorbance value at 445nm with the mixture of test tube No.0
as reference. Taking fucoxanthin as abscissa and absorbance
as ordinate, the regression equation was obtained.

![](https://static.igem.wiki/teams/4688/wiki/table-one.png)

#### Determination of fucoxanthin content

    The extraction and content
determination of fucoxanthin from P.tricornutum were carried
out by organic solvent extraction method. 80mL of P.
tricornutumwas accurately extracted and centrifuged at
5000r·min-1，4℃ for 10min. The supernatant was
discarded,
freeze-dried for 2d, weighed and ground into powder about
0.1g with anhydrous ethanol, alga powder: anhydrous ethanol
=1:40(g·mL1),60 The supernatant was taken and the absorbance
was measured at 445nm wavelength (OD445) by uv
spectrophotometer. The content of fucoxanthin was calculated
by regression equation

Chlorophyll content measurement

1. On the 4th, 6th, 8th and 10th day of cultivation, 2 mL
   algae solution was collected in the EP tube weighed in
   advance, frozen and vacuum dried for 12 h, and the
   corresponding algae weight was obtained by weighing.
2. Each tube was added with 1.5 mL of 95 % ethanol at 4 °C
   for 24 h in the dark with periodic oscillation during
   soaking.
3. After the extraction was completed, the supernatant was
   separated by centrifugation at 4℃, 12000 rpm for 10 min,
   and the supernatant was injected into the microplate.
   The absorbance at 470 nm, 649 nm and 665 nm was read and
   the chlorophyll concentration was calculated according
   to the formula to calculate the pigment content.

Cchla (mg·L-1)= 13.95A665 –
6.88A649

Cchlb (mg·L-1)= 24.96A649 –
7.32A665

Ccarotenoid
(mg·L-1)=(1000A470 – 2.05
Cchla -114 Cchlb)/245

pigment content (mg·g-1DW) = pigment concentration ×
extraction volume（L）/ sample dry weight（g）

Extraction and determination of total carbohydrate from
microalgae

Total carbohydrate content was
quantified using the phenol-sulphuric acid method . About 10
mg of lyophilized algae was resuspended in 1 mL of deionized
water. This algal suspension was added with 1 mL of 5%
phenol solution (w/v) was rapidly added directly to be
mixture. The mixture was incubated for 10 min at room
temperature and then 30 °C for 20 min. The content of
carbohydrates was detected at 483 nm. Glucose was used as
standard at different concentrations (up to 1
g·L−1).

![](https://static.igem.wiki/teams/4688/wiki/table-two.png)

Extraction and determination of total lipids from
microalgae

Total lipid content was also determined by gravimetric
analysis. About 20 mg of lyophilized algae was mixed with 2
mL of chloroform, 2 mL of methanol and 1 mL of 5% NaCl by
vortex for 2 min, then centrifuged at 8000g for 4 min at 10
°C. The chloroform phase was collected and kept for
subsequent analysis. The residual extract was extracted
three more times. All the collected chloroform phase were
mixed together and dried under nitrogen flow. The dried
lipid residue was further dried in oven at 60 °C and the
total lipid content was quantified as a percentage of the
dried weight of algae.

Determination of neutral lipid content in microalgae

1. Take appropriate amount of algae liquid (the same group
   is recommended to do 5 groups of parallel)
2. Add 15% volume fraction of DMSO, add Nile red dye; dark
   20min, during which 3-5min vortex shock increases dyeing
   efficiency.
3. The 300ul sample was added to the enzyme plate, and the
   same volume of PBS was used as the blank control.
4. Parameters detected by enzyme labeling instrument: Read
   Mode: RFU; Wavelengths: Wavelengths 1 Magneto
   ExcitationRod 488nm Emission:592nm, Auto Cutoff

Visualization of microalgal oil bodies

To visualize oil bodies and assess
their morphology, localization, and numbers in the cultured
cells, Nile red and BODIPY 505/515 were applied under dark
conditions in accordance with a similar procedure to that
described for the determination of neutral lipid and TAGs
contents. Mixtures were thoroughly mixed, applied to a glass
slide, covered with a coverslip after 10 min, and then
observed under a Laser scanning confocal microscope LSM
(Leica) (Nile Red: excitation 543 nm, emission 570–610 nm;
BODIPY 505/515: excitation 488 nm, emission 530 nm).
Pictures were randomly acquired from at least 20 cells per
sample, and typical images are presented.

1. Microalgae in logarithmic growth phase ( the fastest
   growing ) were inoculated in a column photobioreactor, 1
   % CO2,23 ° C, 300 μmol photons /
   m2 · s
   continuous
   illumination for 24 h.
2. 2.5 ml of microalgae solution containing about 2 × 106
   microalgae cells was pretreated by microwave oven for 40
   s, 1 % dimethyl sulfoxide ( DMSO ) was added, 75 μl 50
   μg / ml NR ( final concentration was 1.5 μg / ml ) was
   added, 40 ° C was dark, and Nile red ( NR ) dye was used
   for 5 min.
3. 300 × g centrifuged at room temperature for 5 min,
   discard the supernatant.
4. Add 4 ml PBS to the precipitation of microalgae cells,
   mix with a pipette gun, centrifuge at 300 × g for 5 min
   at room temperature, and discard the supernatant.
5. The microalgae cells were resuspended with 2 ml PBS,
   washed, and centrifuged at 300 × g at room temperature
   for 5 min. The supernatant was carefully removed and the
   cell precipitate was collected. Repeat once.
6. The excess fluorescent dye was removed, and 200 μl
   liquid was reserved, which was filtered by 70 μm filter
   membrane and detected by flow cytometry.
7. On a flow cytometer, the detection parameters ( NR
   excitation wavelength of 543 nm, emission wavelength of
   598 nm, fluorescence signal located in FL3 channel )
   were first set with unstained microalgae cell
   suspension, and the negative region was adjusted. At
   least 1 × 105 positive cells were obtained for
   subsequent data analysis.

Determination of photosynthetic index

By using AquaPen AP110, the QY, OJIP and NPQ1 indexes of
algae liquid were measured, which reflected the energy
conversion efficiency of *Phaeodactylum tricornutum* in
photochemical activity.

Microbial embedding

1. Take a certain volume of algal cell concentrate or bacterial
   cell concentrate and pre-sterilized SA (sodium alginate)
   solution 1:1 and mix well to form a mixture. Shaker 180rpm
   21℃ 30min
2. Use the hardware designed by the team or inject it into a
   1ml syringe, at a distance of 15-20cm from the surface of
   the pre-cooled CaCl2, drop into the mixture that
   is the
   formation of 4mm in diameter algal spheres, static fixed at
   room temperature for 2h, rinsed three times with sterile
   water, placed in the seawater with the mother liquor
   culture.

Extraction and determination of total protein from
microalgae

The BCA Protein Assay Kit is based on the BCA method, which is
one of the most commonly used protein concentration assays in
the world, and offers simplicity, stability, sensitivity and
compatibility. The lower limit of detection is 5 μg/ml, and the
minimum amount of protein to be detected is 0.5 μg.

#### Protein standard curve

1. Take an enzyme plate and add reagents as shown below
![](https://static.igem.wiki/teams/4688/wiki/table-three.png)2. According to the number of standard wells, prepare the
   appropriate amount of BCA working solution according to 50
   volumes of BCA reagent A plus 1 volume of BCA reagent B
   (50:1) and mix thoroughly.
3. Add 200 μL of BCA working solution to each well.
4. Place the plate on a shaker for 30sec, leave it at 37℃ for
   30 minutes, and then determine colorimetrically at 562nm.
   Plot the standard curve with the protein content (μg) as the
   horizontal coordinate and the absorbance value as the
   vertical coordinate.

Protein content detection

1. Weigh the appropriate amount of freeze-dried microalgae
   powder into the test tube
2. Add a certain amount of RIPA Lysis Buffer and use a cell
   sonicator for 20 min (40% w, 0.5s on, 2s off)
3. Dilute the sample to be measured to the appropriate
   concentration, making a total volume of 20 μL of sample
   dilution, add 200 μL of BCA working solution, mix
   thoroughly, and place at 37°C for 30 minutes, then use tube
   0 of the standard curve as a reference, colorimetric at 562
   nm, and record the absorbance value.
4. According to the absorbance value of the measured sample,
   the corresponding protein content (μg) can be found on the
   standard curve, divided by the total volume of the sample
   dilution (20 μL), multiplied by the sample dilution times to
   be the actual concentration of the sample (unit: μg/μL).

## DNA

DNA concentration determination

The concentration of 1 μl sample was determined in the DNA
mode of ThermoTM NanoDrop2000/200C.

DNA electrophoresis

Agarose was dissolved into 1X TAE solution (1.2% or 1.5%,
i.e. 1.2g/100ml or 1.5g/100ml) using an electronic
analytical balance, heated in microwave oven to dissolve,
waited for the temperature to drop to about 50~60℃, poured
into the gel version, and pulled off the mold for sampling
after the agarose gel solidified, and selected a suitable
program for electrophoresis.

DNA Purification

1. In a 1.5 ml microcentrifuge tube, add 5 volumes of
   Binding Buffer to 1 volume of PCR products (50-100 μl).
   Mix briefly by vortexing sample.
2. Transfer all the mixture to a provided Spin Column with
   a Collection Tube (to increase the yield of purified
   DNA, incubate for 1 minute).
3. Centrifuge at 10,000 × g for 1 minute. Discard the
   flow-through.
4. Add 650 μl of Wash Buffer to the column. Centrifuge at
   10,000 × g for 1 minute. Discard the flow-through.
5. Centrifuge the empty column at 10,000× g for 1-2 minutes
   to remove any residual Wash Buffer.
6. Place the spin column in a clean microcentrifuge tube,
   add 30-50 μl of Elution Buffer or sterile, distilled
   water (pH >7.0) directly to the center of the column
   matrix (to increase the yield, prewarmed Elution Buffer
   or water can be used). Incubate the column at room
   temperature for 1 minute. Centrifuge at 10,000xg for
   1minute to elute the DNA. The isolated DNA is ready to
   use or can be stored at -20°C.

Plasmid DNA extraction

1. Centrifuge overnight bacterial cell culture (refer to
   above table) at 12000×g for 2 minutes and discard the
   supernatant.
2. Add 5/10 ml of colorless Resuspension Buffer (containing
   RNase A). Mix thoroughly by vortexing.
3. Add 5/10 ml of blue Lysis Buffer, mix by gently
   inverting the tube 4-6 times to completely lyse cells.
   Incubate at room temperature for 5 minutes (lysate
   should change color to blue).
4. Add 7/14 ml of yellow Neutralization Buffer, mix by
   gently inverting the tube 5-6 times. Incubate at room
   temperature for 2 minutes (lysate should change color to
   yellow).
5. Centrifuge at 12,000×g for 15 minutes, gently transfer
   the supernatant to a spin column(if more than 50 ml,
   repeat the transfer after step 6).
6. Centrifuge at 8000×g for 2 minutes and discard the
   flow-through.
7. Add 5 ml of ToxinOut Buffer, incubate at room
   temperature for 10 minutes. Centrifuge at 8,000×g for 2
   minutes and discard the flow-through.
8. Add 3/5 ml of Wash Buffer, centrifuge at 8,000×g for 2
   minutes and discard the flow-through.
9. Repeat step 8 once.
10. Centrifuge at 8,000×g for 5 minutes to completely remove
    the remaining Wash Buffer.
11. Incubate at room temperature for 10 minutes to
    evaporate ethanol.
12. Place the spin column in a clean 50 ml centrifuge tube,
    add 1-2 ml of of EB or sterile, distilled water (pH>7.0)
    directly to the center of the column matrix (for higher
    yield, use prewarmed (60-70℃) Elution Buffer or
    distilled water).
13. Centrifuge the column at 8000×g for 2 minutes to elute
    DNA.
14. Isolated plasmid DNA can be stored at -20℃.

DNA Ligation（infusion）

1. Thaw a vial of Trans1-T1 Phage Resistant Chemically
   Competent Cell on ice.
2. Transfer 2 μl of reaction mixture into 50 μl of
   Trans1-T1 Phage Resistant Chemically Competent Cell and
   mix gently by flicking the tube (do not vortex).
   Incubate on ice for 30 minutes.
3. Heat-shock in a water bath at 42℃ for 30 seconds, and
   immediately place on ice for 2 minutes.
4. Add 450 μl of room-temperature SOC/LB medium. Place in a
   shaking incubator at 37℃ for 1 hour at 250 rpm.
5. Pre-warm LB plate containing the appropriate selection
   antibiotic at 37℃.
6. Spread 100 μl of cells evenly on the selection plate and
   incubate overnight at 37℃.

PCR（pfu ultra enzyme）

1. Add the following components in a pcr tube:
![](https://static.igem.wiki/teams/4688/wiki/table-four.png)2. Perform PCR with thermocycle:
![](https://static.igem.wiki/teams/4688/wiki/table-five.png)

PCR (Taq enzyme)

1. Add the following components in a pcr tube:
![](https://static.igem.wiki/teams/4688/wiki/table-six.png)2. Perform PCR with thermocycle:
![](https://static.igem.wiki/teams/4688/wiki/table-seven.png)

Incision enzyme

FlyCut" Sacl is expressed and purified from E.coli that
carries the recombinant Sacl gene. The molecular weight is
40.8 kDa, with the recognition site at GAGCT^C. The reaction
is conducted for 5- 15 minutes at 37°C, and heat-inactivated
at 80℃ for 20 minutes. This enzyme is not sensitive to dam,
dcm or mammalian CpG methylation.

![](https://static.igem.wiki/teams/4688/wiki/table-eight.png)

FlyCut Xhol is expressed and purified from E.coli that
carries the recombinant Xhol gene. The molecular weight
is27.9 kDa, with the recognition site at C^TCGAG. The
reaction is conducted for 5- 15 minutes at 37°C, and
heat-inactivated at 65°℃ for 20 minutes. This enzyme is not
sensitive to dam or dcm methylation, but sensitive to
mammalian CpG methylation.

![](https://static.igem.wiki/teams/4688/wiki/table-nine.png)

Transformation of chemically competent cells

1. Equilibrate a water bath to 42℃.
2. Warm a vial of SOC medium or LB medium to room
   temperature. Warm selective plates at 37°C for 30
   minutes.
3. Thaw a vial of 100 μl of Trans1-T1 Phage Resistant
   Chemically Competent Cell on ice, aliquot 50 μl of the
   cells into a prechilled
4. 1.5 ml tube, add target DNA (1 to 5 μl) into the tube.
   Do not mix by pipetting up and down. Incubate the cells
   on ice for 30 minutes.
5. Heat-shock the cells for 30 seconds at 42°C without
   shaking. Immediately transfer the tube to ice.
   6.Incubate on ice for 2 minutes without shaking.
6. Add 500 μl of prewarmed SOC medium or LB medium (without
   antibiotic) into the tube, mix well and shake at 37℃
   for 1 hour at 200 rpm for cell recovery and for the
   expression of antibiotic resistance.
7. Spread 20 to 200 μl from each transformation vial on a
   prewarmed selective plate. The remaining can be stored
   at 2-8℃ and plated the next day if needed.
8. Invert the plate and incubate at 37°C overnight.
9. Select colonies and analyze by restriction enzyme
   digestion, PCR, or sequencing.

## RNA

Validation using real-time PCR（TRANSTM）

To further validate the RNA-Seq results, RNA extracted from
the same culture for RNA-Seq was subjected to the
PrimeScript RT reagent kit with gDNA Eraser for cDNA
synthesis. qPCR was performed using standard methods as
previously described . β-Actin was used as a housekeeping
marker. Primer pairs used for qPCR analysis are listed in
attachment.

Total RNA extraction & reverse transcription

1. The wild-type *Phaeodactylum tricornutum* was collected by
   low-temperature centrifugation in the enzyme-free EP tube,
   and the precipitate was washed three times with DEPC water
   and placed in an ultra-low temperature refrigerator at-80 ℃
   for storage.
2. The algae samples were fully ground in a mortar baked at 180
   C for more than 4 h, and the algae powder was divided into
   non-enzymatic EP tubes, and 1 mL Trizol Reagent was added to
   each EP tube, and the samples were fully dispersed by vortex
   ; stand on ice for 3-5 min.
3. Add 200 1μL chloroform to the lysis buffer according to 1
   mL Trizol Reagent, shake sharply with hands for 15 s, stand
   on ice, centrifuge at 12,000 × g, transfer the supernatant,
   add equal volume of isopropanol to stand on ice to
   precipitate RNA, centrifuge at 12,000 × g, remove the
   supernatant, add 75 % ethanol to repeat washing for 2-3
   times, and leave for 1 minute.
4. the lid was placed in a fume hood for air drying for 10-15
   min, and the RNA was dissolved after the ethanol in the EP
   tube was volatilized.3 1μL RNA was taken for agarose gel
   electrophoresis to detect the quality, and the RNA
   concentration was measured for inversion.

On ice, the reaction mixture was prepared
according to the following table and reacted at 42 ° C for 30
min.

![](https://static.igem.wiki/teams/4688/wiki/table-ten.png)

## Protein

Protein extraction

1. Leach the algae ball and freeze it with liquid nitrogen,
   grinding into powder, packed in 1.5 ml EP tube.
2. Add 1 ~ 2.5 times of NG buffer to the fume hood, and mix
   well on the vortex meter.
3. Heating on a 100 °C thermostat for 10 min.
4. 12,000 rpm Centrifugation for 10 min.
5. Take the supernatant for SDS-PAGE electrophoresis
   （step1：86V， 30min；step2：130V，1.5h）.

Western blot

1. After the electrophoresis, cut off the protein gel
   block, place it in a protein semi-dry transfer
   instrument, and transfer the target protein to the PVDF
   membrane under the condition of 15 V 13 min.
2. Put the PVDF membrane into 50 g/L skimmed milk powdr
   shake).
3. Use 20 g/L skimmed milk powder to dilute the anti-His
   mouse antibody at a ratio of 12000, and add an
   appropriate amount of the diluted antibody.
4. Submerge the PVDF membrane in a self-made closed bag
   containing PVDF membrane, and incubate overnight at 4 'C
   (slowly shaking).
5. Take out the PVDF mem-brane from the primary antibody
   solution and place it in a petri dish, wash with TBST 5
   times, each time 15min.
6. Use 20 g/L skimmed milk powder to dilute goat anti-mouse
   lgG (called secondary antibody) at a ratio of 1:3000,
   and add an appropriate amount of diluted secondary
   antibody to a self-made closed bag with PVDF membrane to
   submerge the PVDF membrane and incubated at room
   temperature for 2 hours(slowly shake).
7. take out the PVDF membrane from the secondary antibody
   solution and place it in a plate, wash five times with
   TBST, 15 minutes each time.
8. After washing. dry the PVDF membrane and drop it on the
   front. Add the prepared color developer until it is
   completely covered. place it at room temperature for 2
   minutes, place it in an ECL luminescence imager for
   exposure and photographing. and record the result.

{% 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

↓

---

[Overview](#overview)

[Part I](#Result1)

[Part II](#Result2)

[Part III](#Result3)

[Part IV](#Result4)

[Summary](#Summary)

[Additional Product](#Additional performance)

#### Overview

Our project aims to investigate the potential of utilizing engineered microalgae strains and microalgae-bacteria interaction systems for water remediation. Firstly, we optimized the efficiency of the microalgae-bacteria interaction system by screening different ratios of *P. tricornutum* and *Q. aquimaris*. Subsequently, we compared various immobilization methods to enhance the stability and repeatability of the microalgae-bacteria system. Furthermore, through synthetic biology techniques, we constructed engineered microalgae strain ——FUCO3 with enhanced nitrogen absorption, lipid accumulation, and fucoxanthin accumulation, which were then screened and filtrated. Finally, we evaluated the bioremediation effectiveness of these engineered strains under varying water quality conditions and analyzed their impact on aquatic environments. This study provides a new bioremediation idea and method for solving water pollution problems using engineered microalgae strains and microalgae-bacteria interaction systems.

![](https://static.igem.wiki/teams/4688/wiki/pp1.png)

![](https://static.igem.wiki/teams/4688/wiki/fg2.png)

#### Screening the optimal ratio for the interaction of *P. tricornutum* and *Q. aquimaris*

##### Introduction

*Phaeodactylum tricornutum* is an economically significant microalga in marine ecosystems. It serves as both aquaculture feed and a source of valuable bioactive compounds such as EPA, DHA, and fucoxanthin. *Qipengyuania aquimaris*, a widely distributed bacterium found in marine environments, exhibits functions including organic matter degradation and heavy metals detoxification. The interaction between *P. tricornutum* and *Q. aquimaris* holds great potential for bioremediation strategies aimed at effectively addressing issues related to nitrogen-phosphorus elements, heavy metals, and organic pollutants in marine ecosystems. However, the molecular mechanisms and regulatory factors governing this interaction remain unclear; furthermore, the physiological effects of different bacteria-to-microalgae ratios on microalgae require further research and exploration.

##### Results

In order to explore the optimal ratio of microalgae-bacteria interaction between *P. tricornutum* and *Q. aquimaris*, we used the cell density ratio of the two as the basis, and set up four treatment groups with microalgae-bacteria ratios of 1:1, 1:3, 1:5, and 1:7, respectively, to monitor physiological indicators. The results showed that in the interaction system with *Q. aquimaris*, the cell growth rate increased significantly with the increase of bacteria concentration, and the biomass increased significantly in the 1:3 and 1:5 treatment groups (Figure 1 A,B), but the photosynthetic efficiency Fv/Fm decreased significantly (Figure 1 C). In addition, combined with the analysis of chlorophyll and fucoxanthin relative content (Figure 1 C,D), it was found that the accumulation of microalgal pigments had a significant positive effect within a certain range with the increase of bacteria concentration.

Additionally, by conducting visual analysis of the microalgae oil plasts and analyzing the average fluorescence intensity of the oil plasts using flow cytometry (Figure 2), it was observed that the inclusion of *Q. aquimaris* in the culture enhanced lipid accumulation in microalgae to a certain extent when compared to the control.

![](https://static.igem.wiki/teams/4688/wiki/result-all/p2.png)

**Figure 1 Effects of different microalgae-bacteria ratios on microalgae related indicators**

(A, cell growth curve; B) biomass; C, photosynthetic efficiency Fv/Fm; D, relative chlorophyll content; E, relative content of fucoxanthin; microalgae: bacteria is cell density ratio.)

![](https://static.igem.wiki/teams/4688/wiki/result-all/p3.png)

**Figure 2 Visualization and mean fluorescence intensity analysis of microalgal oil plasts**

##### Conclusions

By culturing P.tricornutum with *Q. aquimaris* at various density ratios, we assessed the growth rate, biomass, oil accumulation, photosynthetic efficiency, and pigment accumulation of P.tricornutum. The findings revealed that:

**·**The addition of *Q. aquimaris* has a more obvious improvement on the growth rate of *P. tricornutum*, and has a significant promotion effect on biomass.

**·**As the concentration of *Q. aquimaris* increased in the co-culture system, the photosynthetic efficiency of P.tricornutum initially increased rapidly and then gradually .The treatment groups with the ratios of 1:1 and 1:3 exhibited significant improvement in Fv/Fm on D1, while the group with a ratio of 1:3 maintained good photosynthetic efficiency until D5.

**·**Relative pigment content demonstrated that treatment groups with ratios of 1:1 and 1:3 promoted pigment accumulation in P.tricornutum approximately 1.3-fold respectively; However, treatment group with a ratio of 1:7 exerted a significant inhibition on chlorophyll content consistent with poor photosynthetic efficiency.

Regarding lipid accumulation in the interaction system between P.tricornutum and *Q. aquimaris* , there was a substantial elevation in lipid accumulation within the groups with ratios of 1:1 and 1:3. Specifically, lipid accumulation increased by 80.08% compared to the control for the 1:3 treatment group.

![](https://static.igem.wiki/teams/4688/wiki/result-all/p4.png)

Based on these findings, we primarily analyzed photosthetic efficiency and lipid accumulation as key factors to evaluate bioremediation performance in co-cultivation systems. Consequently, we selected a co-cultivation ratio of *P. tricornutum* to *Q. aquimaris* at of 1:3 for further experiments.

##### Prospect

These findings suggest a mutualistic relationship between *P. tricornutum* and *Q. aquimaris*, potentially involving the exchange of nutrients, signaling molecules, and antioxidants. The results of this study offer an effective co-culturing method for marine microalgae and bacteria with significant implications for the development of microalgae-based biofixation, biodiesel and functional pigments synthesis

![](https://static.igem.wiki/teams/4688/wiki/fg2.png)

#### Comparing different immobilization schemes for microalgae-bacteria interaction system

##### Introduce

Microalgae-bacteria interaction can be carried out by symbiotic cultivation or separate cultivation. Symbiotic cultivation means that microalgae and bacteria or fungi grow together in the same medium, while separate cultivation means that microalgae and bacteria or fungi grow separately in different media, and then are mixed by physical or chemical methods. Different cultivation methods will affect the effect and mechanism of microalgae-bacteria interaction.

##### Results

In order to further analyze the interaction mechanism of *P. tricornutum* and *Q. aquimaris* and screen out a relatively optimal immobilization cultivation mode, we designed five interaction cultivation forms under the premise of the selected microalgae-bacteria ratio.

![](https://static.igem.wiki/teams/4688/wiki/result-all/p5.png)

By monitoring the nitrogen consumption rate in the medium (Figure 3 A), we found that the ABO treatment group had the fastest nitrogen absorption rate compared with the conventional interaction form of AB control group, and AO and ABS had relatively slower nitrogen absorption rates. According to the above phenomenon, it can be seen that when microalgae are isolated by embedding materials, although they are still in the same medium with bacteria, their influence by the substances produced by bacteria is significantly enhanced, but when microalgae and bacteria are mixed and co-embedded (ABS), this phenomenon does not occur.

Then, by comparing the fucoxanthin (Figure 3 B) and chlorophyll (Figure 3 C) of each treatment group, it was found that when bacteria were immobilized in one of the forms in the treatment group, they had a positive effect on the accumulation of chlorophyll and fucoxanthin. In addition, by oil plasts visualization analysis (Figure 4), it was found that when bacteria were separately embedded and immobilized, the lipid accumulation of microalgae in the same environment was significantly reduced, but the volume and fluorescence intensity of microalgal chloroplasts increased.

![](https://static.igem.wiki/teams/4688/wiki/result-all/p6.png)

**Figure 3 The effect of different embedding conditions on microalgae**

(A, the change of total nitrogen content in the medium; B, the relative chlorophyll content of different treatment groups; C, the relative fucoxanthin content of different treatment groups. microalgae-bacteria liquid co-culture (AB), microalgae (AO) or bacteria (BO) separately immobilized and co-cultured with bacteria or microalgae, microalgae and bacteria separately immobilized and co-cultured (ABO), microalgae and bacteria co-immobilized (ABS); chlorophyll and fucoxanthin detection for D7)

![](https://static.igem.wiki/teams/4688/wiki/result-all/p7.png)

**Figure 4 TAGs accumulation in microalgae**

（TAGs content was determined by BODIPY 505/515 Laser scanning confocal microscope LSM (Leica),with 488 nm excitation wavelength and 530 nm emission wavelength）

##### Conclusions

It can be seen from the above analysis that the effect of different embedding conditions on microalgae is very significant. Considering the convenience of microalgae collection and the good nitrogen removal efficiency and pigment accumulation, we suggest using the interaction mode of single immobilized bacteria (BO) for cultivation; for those who need to significantly increase the fucoxanthin production, we suggest using the scheme of microalgae-bacteria interaction after co-immobilizing bacteria and microalgae (ABS).

##### Prospect

**·** The study provides an effective microalgae-bacteria interaction mode (BO) for microalgae immobilization cultivation technology, which can achieve efficient nitrogen removal and fucoxanthin accumulation at the same time. This is of great significance for using microalgae to treat nitrogen-containing wastewater and extract high-value pigments.

**·** The study provides a simple and feasible embedding method for microalgae immobilization cultivation technology, which can use low-cost materials (such as agar, gelatin, etc.) to prepare carriers suitable for microalgae growth and metabolism. This has a positive effect on reducing the input cost of microalgae immobilization cultivation technology and promoting its scale-up application.

**·** The study provides a systematic evaluation method for microalgae immobilization cultivation technology, which can comprehensively consider the effects of different embedding conditions on microalgae growth, metabolism, collection, etc. This has guiding significance for optimizing the parameter setting and process flow of microalgae immobilization cultivation technology.

![](https://static.igem.wiki/teams/4688/wiki/fg4.png)

#### Filtrating of the engineered microalgae strains

By designing corresponding primers, we successfully amplified and cloned BBa\_K4688002 to BBa\_K4688004 from the *P. tricornutum* genome. Then, we used the infusion method to connect the obtained fragments to the expression vector provided by the 2022 CHINA-FAFU team, and successfully identified them (Engineering-Design Cycle 3: Test).

We transformed the constructed expression vector into *P. tricornutum* by electroporation, and obtained potential engineered microalgae strain monoclonal clones on the selection plate with zeocin resistance after 15 days (Figure 5 A); then, we randomly picked monoclonal microalgae strains and cultured them in seawater with resistant medium, and verified the picked monoclonal clones at DNA, RNA, and Protein levels respectively (Figure 5 B,C,D).

At the Protein level verification (Figure 5 B), we took the monoclonal microalgae strains that had been enlarged and cultured, and used the tag in the expression vector to perform Western Blot verification. The results showed that only FUCO3 engineered microalgae strain had a protein size that matched the expectation (FUCO3: 84.15kDa), while FUCO1 (81.51 kDa) and FUCO2 (84.16 kDa) results were much larger than the expected protein band size.

In order to further confirm the correctness of the engineered microalgae strain, we verified it at the DNA level (Figure 5 C), using the positive control of the expression vector that was verified correctly in the previous stage as a reference, and using the universal identification primers designed with 50bp before and after the gene insertion site in the expression vector to perform PCR amplification on DNA; by comparison, we successfully identified FUCO1 and FUCO3 engineered microalgae strains. However, since FUCO1 and FUCO2 had similar results that were too large at the protein level, and FUCO2 microalgae strain did not pass the identification at the DNA level, we chose to perform a new round of microalgae transformation on FUCO1 and FUCO2, and temporarily continued with FUCO3 microalgae strain for subsequent identification.

In addition, in order to analyze whether the expression vector successfully overexpressed the target gene in microalgae, we performed RNA level identification on FUCO3 (Figure 5 D), and obtained positive results. The expression level of *PtFUCO3* gene in FUCO3 microalgae strain was about 8-fold higher than that in WT at the same period.

Finally, after multiple identifications, we selected FUCO3 engineered microalgae strain to carry out subsequent experimental plans.

![](https://static.igem.wiki/teams/4688/wiki/result-all/p8.png)

**Figure 5 Identification of the engineered microalgae strain**

(A, monoclonal transgenic microalgae strain; B, protein level identification; C, DNA level identification; D, RNA transcription level identification)

![](https://static.igem.wiki/teams/4688/wiki/fg3.png)

#### Evaluating the bioremediation potential of genetically modified microalgae strain

##### Introduce

Based on the previous results of the team, the bioremediation capacity of the successfully identified FUCO3 engineered microalgae strain was evaluated, and the microalgae-bacteria interaction system (WT-Qa, FUCO3-Qa treatment group) was added to further enhance its remediation capacity.

##### Results

In terms of cell growth rate and water nitrogen utilization, we found that FUCO3 microalgae strain had a higher growth rate in the early stage of the growth cycle (Figure 6 A), and reached a similar level to the control in the later stage; in terms of nitrogen utilization efficiency, FUCO3 microalgae strain had a faster nitrogen utilization efficiency than the control (Figure 6 B), and achieved a more efficient nitrogen removal after forming an microalgae-bacteria interaction system with *Q. aquimaris*.

![](https://static.igem.wiki/teams/4688/wiki/result-all/p9.png)

**Figure 6 The monitoring of microalgae growth and nitrogen use efficiency**

（WT，wild type；FUCO3，Overexpressing strain of *PtFUCO3*；WT-Qa：Bacteria and microalgae interactions of wild type microalgae strains；FUCO3-Qa，Bacteria and microalgae interactions in microalgae strains overexpressing *PtFUCO3*）

In terms of photosynthetic efficiency and pigment accumulation, analysis of the Fv/Fm measurement results revealed that the engineered microalgae strain FUCO3 exhibited significantly higher photosynthetic efficiency during the D1 adaptation period of the growth cycle (Figure 7 A). This enhancement could potentially be attributed to increased levels of antioxidant substances such as fucoidan within its microalgae, thereby considerably shortening the adaptation period for this engineered strain. Furthermore, examination of NPQ results pertaining to light energy dissipation capacity demonstrated that FUCO3 displayed superior photosynthetic resistance under both conventional cultivation and microalgae-bacteria interaction cultivation conditions. Compared to WT, FUCO3 exhibited an approximate 35% increase in both photosynthetic resistance and high light (Figure 7 B). In terms of microalgal pigment accumulation, FUCO3 exhibits a significant improvement compared to the wild type, with an approximately 1.75-fold increase in fucoxanthin content (Figure 7 C).

![](https://static.igem.wiki/teams/4688/wiki/result-all/p10.png)

**Figure 7 Measurement of microalgal photosynthetic capacity and pigment accumulation**

(A, photosynthetic efficiency Fv/Fm; B, relative content of fucoxanthin; C, photosynthetic NPQ level)

Regarding lipid accumulation, through visualization of microalgal oil bodies and analysis of average fluorescence intensity during the D7 cultivation cycle, we observed that the FUCO3 microalgae strain exhibited a 40% increase in lipid accumulation under conventional cultivation conditions and a 22% increase under microalgae-bacteria interaction mode compared to the control (Figure 8).

![](https://static.igem.wiki/teams/4688/wiki/result-all/p11.png)

**Figure 8 Visualization analysis and mean fluorescence intensity analysis of microalgal oil plasts**

In addition, in order to further test the ability of engineered microalgae and the designed bacteria-microalgae co-culture system in wastewater bioremediation, we used simulated artificial wastewater for testing in the laboratory. Since the expectation for wastewater bioremediation in the real society is fast enough and takes into account the effect, we focus on the early data of the engineered microalgae in the test phase. Through analysis, it can be found that in the artificial wastewater environment, the cell growth rate, biomass and photosynthetic efficiency of FUCO3 microalgae are significantly higher than those of the control (Figure 9 A, B,C). Moreover, analysis of cell growth rates indicated that incorporation of the bacteria-microalgae co-culture system conferred a certain level of resistance to wild-type *P. tricornutum* in simulated artificial wastewater conditions (Figure 9 A).

![](https://static.igem.wiki/teams/4688/wiki/result-all/ppp.png)

**Figure 9 Physiological monitoring of microalgae under artificial wastewater conditions**

(A, cell growth curve; B, Biomass; C, photosynthetic efficiency Fv/Fm; WT: wild type; FUCO3, *PtFUCO3* overexpressing engineered microalgae; WT-W & FUCO3-W, two microalgae cultured under simulated wastewater conditions; WT-W-Qa & FUCO3-W-Qa: two microalgae co-cultured with bacteria under simulated wastewater conditions)

Finally, we evaluated the expression levels of key genes, fucoxanthin synthesis, and nitrogen and phosphorus utilization pathways in FUCO3 microalgae, which possess superior characteristics such as enhanced lipid accumulation, removal of eutrophication-causing elements, and improved photosynthetic efficiency. Encouragingly, a significant up-regulation trend was observed for most of the key genes in these pathways (Figure 10), providing robust molecular evidence to support our subsequent analysis on the role of *PtFUCO3* in related metabolic processes.

![](https://static.igem.wiki/teams/4688/wiki/result-all/p13.png)

**Figure 10 Analysis of the key gene expression in different metabolic pathways**

##### Conclusions

This study constructed FUCO3 engineered microalgae strain and explored its characteristics in water nitrogen utilization, photosynthetic efficiency, pigment accumulation and lipid accumulation, and compared it with the control WT. The results showed that FUCO3 engineered microalgae strain had higher cell growth rate, nitrogen utilization efficiency, photosynthetic efficiency, high light response ability and lipid accumulation, and the key genes in the related metabolic pathways showed a significant up-regulation trend.

It provided a molecular basis for further revealing the role of *PtFUCO3* in the related metabolic pathways, and also provided new ideas and candidate varieties for using microalgae for water purification and bioenergy production.

#### Prospect

This study firstly introduced the fucoidan hydrolysis gene *PtFUCO3* into the *P. tricornutum*, and constructed the engineered microalgae strain FUCO3 with high nitrogen utilization and lipid accumulation capacity, and systematically analyzed its characteristics in water nitrogen utilization, photosynthetic efficiency, pigment accumulation and lipid accumulation, revealing the possible role of *PtFUCO3* in the related metabolic pathways.

It not only provided new ideas and methods for deep understanding of the synthesis and regulation mechanism of fucoxanthin, but also provided new ideas and candidate varieties for using microalgae for water purification and bioenergy production. However, there are some limitations and shortcomings in the study, such as FUCO3 engineered microalgae strain did not show obvious advantages in pigment accumulation, which might be related to the competition or inhibition between fucoxanthin and other pigments such as chlorophyll; in addition, the study only analyzed the role of *PtFUCO3* in the related metabolic pathways from the gene expression level, and failed to verify it from the enzyme activity and metabolite level.

Therefore, future research can be expanded from the following aspects: first, further explore the interaction and regulation mechanism between fucoxanthin and other pigments, and optimize the pigment accumulation capacity of the engineered microalgae strain; second, use proteomics and metabolomics techniques to verify the role of *PtFUCO3* in the related metabolic pathways from multiple levels and dimensions; third, evaluate the stability and adaptability of FUCO3 engineered microalgae strain under different cultivation conditions and application scenarios, and develop more application products and services. This study has important social significance and practical value for promoting the development of microalgae biotechnology, improving the efficiency and benefit of water purification and bioenergy production.

### Summary

In our project, we introduced a study on the co-culture of *P. tricornutum* and *Q. aquimaris*, and an experiment of overexpressing fucoidan synthesis related genes in *P. tricornutum* by genetic engineering. The results showed that the co-culture of bacteria and microalgae can improve the growth rate, biomass, lipid accumulation, photosynthetic efficiency and pigment accumulation of microalgae, and FUCO3 engineered microalgae showed better performance than wild-type *P. tricornutum* in these aspects, and also showed great potential in wastewater bioremediation. These findings revealed the important role of bacterial-algal interaction and fucoidan synthesis genes in microalgal metabolism, and provided new ideas and methods for the application of microalgae and bacterial-algal co-culture systems.

![](https://static.igem.wiki/teams/4688/wiki/fg2.png)

### Additional Product

#### The compilation of the "Bacteria and Algae manual"

Bacteria-microalgae symbiosis is a mutually beneficial relationship that holds significant potential for applications in bioenergy, biopharmaceuticals, and other fields. However, the knowledge and technical applications of bacteria-microalgae symbiosis are not yet widespread or comprehensive enough. Many beginners who wish to enter this field face the challenge of lacking systematic learning materials and guidance. To address this issue, we have authored a handbook on bacteria-microalgae symbiosis that aims to summarize the fundamental knowledge and practical methods of this topic while facilitating quick initiation into related research for beginners. In writing this handbook, we drew upon relevant literature from both domestic and international sources as well as our own research experience in the field of bacteria-microalgae symbiosis. Our goal was to provide comprehensive yet concise content with clear structure and rigorous logic. We hope that our handbook will serve as a valuable resource for those new to the field while also providing reference material for researchers already working in it.

**The manual serves as a comprehensive guide to the applications of fungal-microalgae interactions, specifically designed to facilitate beginners in swiftly initiating their research endeavors within this field.**

**The manual covers the following content:**

**·**The definition, characteristics and classification of microalgae and bacteria, as well as the morphological structure and physiological features of common microalgae and bacteria;

**·**The application of microalgae and bacteria in various fields, such as water purification, bioenergy, biopharmaceuticals, etc.;

**·**The cultivation methods of microalgae and bacteria and the protection and management measures;

**·**The principle, mechanism, effect and advantage of microalgae-bacteria interaction.

**The manual has the following characteristics**

**·**The content is comprehensive, encompassing the fundamental knowledge, technical methodologies, and practical applications of microalgae-bacteria interaction.

**·**The language is concise yet informative, accompanied by abundant illustrations to facilitate comprehension and implementation.

**·**The structure exhibits clarity and the logical flow maintains rigor, rendering it convenient for both reference and learning purposes.

**The manual holds the following significance**

**·**It offers a comprehensive learning resource for novices in the field of microalgae-bacteria interaction, enabling them to acquire essential knowledge and skills.

**·**It functions as an informative guidebook for promoters involved in the field of microalgae-bacteria interaction, effectively showcasing their accomplishments and demonstrating the significance of this research area.

##### 《Bacteria and Algae manual》

{% endblock %}