iGEM 2022 — Team china-fafu

{% 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 lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

### Engineering

#### Overview

In our project, we focused on enhancing the environmental management ability of *Phaeodactylum
tricornutum* by
constructing five new parts and successfully recombined these five parts into plasmid vectors to construct an
overexpression system. The overexpression system was then transferred to *Phaeodactylum
tricornutum* for
functional validation. Finally, the transformation results were tested and validated to investigate whether the
overexpression system was successfully expressed in algae strains, the effect on its growth and metabolism, and
whether its carbon sequestration capacity was enhanced.

![](https://static.igem.wiki/teams/4112/wiki/lab/engineering/fig1-flow-chart.png)

Fig1. Flow Chart

#### Design

At the beginning of the project, we wanted to use *Phaeodactylum tricornutum* to treat waste
water.
Associating
its strong carbon sequestration ability, we wanted to construct engineered algal strains with stronger
environmental management ability to help mitigate climate problems. After reviewing the documents, we targeted
the following genes to try to construct a recombinant plasmid system.

#### Design Cycle 1：Gene acquisition and expression vector construction

**1. Gene acquisition:** Using the cDNA of P. tricornutum as a template, the *PTCA*-encoded carbonic
anhydrase
of P. tricornutum was cloned by overlap PCR. As a member of the *β-CA* family, it can reversibly catalyze
the CO2
hydration reaction, which can improve CO2 conversion efficiency and play a key role in CCM, as well
as in ion
exchange, CO2 acquisition, and photosynthesis.

*PTCA2*, which is highly homologous to *PTCA*, encodes a protein with fully
conserved *β-CA*
zinc
coordination
residues and significant levels of mRNA accumulation at low CO2 concentrations.

*PRK* encodes phospho-ribulose kinase, an enzyme specific to the Calvin cycle, which plays a
crucial role
in
regulating the flow of sugars in the Calvin cycle by catalyzing the formation of the receptor
ribulose-1,5-bisphosphate of CO2 with the help of ATP phosphorylation.

![](https://static.igem.wiki/teams/4112/wiki/lab/engineering/fig2-electrophoresis-image-of-the-target-gene.png)

Fig2. Electrophoresis image of the target gene

**2. Ligation:** The vector we used is pPink-HC-FHG-cp. pPink-HC-FHG-cp is 8678bp in length and
contains the
Paox1
promoter and CYC1 terminator. We used T4 ligase to ligate *CA*, *CA2*, *PRK* ,*CA-PRK* and
*CA2-PRK* to the vector,
respectively.

![](https://static.igem.wiki/teams/4112/wiki/project/engineering/th-fig3-overexpression-system.png)

Fig3. Overexpression system

At the beginning, we did not insert eGFP tags in the overexpression system, but after talking with experts and
reviewing documents, we decided to insert eGFP for labeling to have a better observation and verification of the
transfer and expression of our recombinant plasmids.

#### Design Cycle 2：Obtaining transgenic strains

![](https://static.igem.wiki/teams/4112/wiki/lab/engineering/fig4-a-ca-prk-overexpression-system-b-ca2-prk-overexpression-system.png)

Fig4. A *CA-PRK* overexpression system. B *CA2-PRK* overexpression system.

**1.Transformation:** The constructed plasmids are transferred into receptor cells for culture.

**2.Validation:** Pick off single colonies for incubation and PCR, select the correct bands for
agarose gel
electrophoresis and sequencing.

![](https://static.igem.wiki/teams/4112/wiki/team/5.png)

Fig5. Colony PCR validation

**3. Plasmid acquisition:** Based on the sequencing results, the culture is expanded, the plasmid
is extracted, and
the plasmid is verified to contain the target gene by PCR assay.

#### Design Cycle 3：Obtaining engineered algae strains

**1.Electroporation:** use electroporation protocol to transfer the plasmid into *Phaeodactylum tricornutum.*

![](https://static.igem.wiki/teams/4112/wiki/lab/engineering/fig-6-implementation-of-transgenic-algae-strain-on-culture-medium.png)

Fig6. Transgenic algae strain screening

**2.Validation:** After screening and culturing, extract the genomic DNA of *Phaeodactylum tricornutum*
and
determine whether the recombinant plasmid is successfully transferred into the algal cells by PCR.

![](https://static.igem.wiki/teams/4112/wiki/lab/engineering/fig-7-dna-pcr-validation-agarose-gel-electrophoresis-results.png)

Fig7. DNA level validation

Extract proteins to verify algal plant function from the protein level.

![](https://static.igem.wiki/teams/4112/wiki/lab/engineering/fig-8-coomassie-bright-blue-stain.png)

Fig8. Coomassie bright blue stain

![](https://static.igem.wiki/teams/4112/wiki/lab/engineering/fig-9-western-blot-verify.png)

Fig9. Western Blot verify

**Target gene expression validation**

![](https://static.igem.wiki/teams/4112/wiki/project/engineering/th-fig10-histogram-of-gene-level-expression-of-target-genes.png)

Fig10. Gene level expression of target genes

##### Design Cycle 4: Seawater culture

We used seawater f/2 medium to cultivate transgenic algae strains, and tested many physiological and
biochemical indicators. The test results are as follows.

##### Cell density

In terms of growth, the growth of the algae strains transferred into the overexpression vector system was not
affected, and there was no significant difference between their cell density and that of wild type algae
strains.

![](https://static.igem.wiki/teams/4112/wiki/lab/engineering/fig11-cell-density-line-graph.png)

Fig11.Cell density

**Chlorophyll fluorescence parameters**

![](https://static.igem.wiki/teams/4112/wiki/lab/engineering/fig12-histogram-of-photosynthetic-rate-on-day-5-of-incubation.png)

Fig12.Photosynthetic rate on day 5 of incubation

**Lipid content and Lipid composition**

![](https://static.igem.wiki/teams/4112/wiki/lab/lsm-1.png)

Fig13. Transgenic microalgae screened for antibiotics

After testing, both transgenic algae strains were found to have significantly higher oil content.
Pha::CA-PRK-eGFP has 2.3 times the oil content of the wild type.

![](https://static.igem.wiki/teams/4112/wiki/team/fig14.png)

Fig14. Fatty acid composition analysis

![](https://static.igem.wiki/teams/4112/wiki/lab/engineering/fig15-folding-graph-of-the-remaining-content-of-nitrogen-and-phosphorus-elements.png)

Fig15. Nitrogen and phosphorus content

##### Design Cycle 5: Validation of gene expression levels

The results showed that the expression levels of genes related to phosphorus transport and lipid metabolism
were significantly up-regulated in one of the transgenic algae strains (Pha:: CA-PRK-eGFP).

![](https://static.igem.wiki/teams/4112/wiki/lab/engineering/fig16-qpcr-analysis-of-key-genes.png)

Fig16. qPCR analysis of key genes

##### Design Cycle 6: Wastewater culture

Based on the above indexes, we screened out a better algal strain (Pha:: CA-PRK-EGFP) for
waste water
culture to
verify its viability in wastewater and nitrogen and phosphorus removal capacity.

After testing, we found that the growth of transgenic algae was not affected under the waste water culture
environment, and the accumulation of lipid was significantly increased.

![](https://static.igem.wiki/teams/4112/wiki/lab/engineering/fig17-cell-density-line-graph.png)

Fig17. Cell density

![](https://static.igem.wiki/teams/4112/wiki/lab/engineering/fig18-nile-red-fluorescence-content-line-graph.png)

Fig18. Nile red fluorescence content

{% endblock %}

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

### Experiments

### 1 General

#### 1.1 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.

#### 1.2 Agar plate

##### 1.2.1 LB- Agar plate

1. Place 10g Tnypxone, 5g Yeast extract, 10g NaCl, 15g Ager, 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℃.

#### 1.2.2 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
ZnSO4-7H2 O, 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℃.

#### 1.3 Liquid medium

##### 1.3.1 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℃.

##### 1.3.2 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℃.

##### 1.3.3 Artificial sewage

Formula (1 ×): NaAC 0.385g/L NH4Cl 0.115g/L MgSO4·7H20 0.066g/L
KH2PO4
0.0130g/L CaCl2
0.006g/L
FeSO4·7H2O
0.00055g/L

#### 1.4 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.

### 2 Microalgae

#### 2.1 Algal species and culture

##### 2.1.1 Algal species

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

##### 2.1.2 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-2s-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.

#### 2.2 Microalgae cell count and growth curve determination

##### 2.2.1 Microalgae cell count

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

##### 2.2.2 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 .

##### 2.2.3 Microalgae 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 Brown Finger Algae triangle culture
cycle.

#### 2.3 Microalgae cell enrichment

Enrichment of 500 ml of algal 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.

#### 2.4 Microalgae grinding

Liquid nitrogen is poured into the mortar to pre-cool the mortar and mortar rod. The enriched algal 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.

#### 2.5 Extraction and determination of Fucoxanthin from microalgae

##### 2.5.1 Fucoxanthin standard curve

Take 11 test tubes, number them, add various solutions according to Table 2, 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/4112/wiki/project/experiments/experiments-pic-1.png)

Fig7.Folding graph of the remaining content of nitrogen and phosphorus elements

##### 2.5.2 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-14 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

#### 2.6 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 10 μg/ml, and the minimum amount of protein to be detected is 0.2 μg. In this experiment,
total protein was extracted and measured using the BCA Protein Quantification Kit BCA protein concentration
assay kit (enhanced version 20201es76).

##### 2.6.1 Configuration of standards and working solutions

1.Take an enzyme plate and add reagents as shown below

1. Formulation of BSA standard system

Standard dilutions are solubilizations of protein samples, in principle in what solution the protein sample is
in, and in what solution the standard is also diluted appropriately. But also available in 0.9% NaCl or 1 × PBS
for dilution.

2. Formulation of BCA working solution

1) Calculate the total BCA working fluid volume required.

Total BCA working fluid volume = (Standard + sample to be tested) × Number of repeats × BCA working solution
required for each sample.

2) Prepare BCA working solution: 50 volumes of BCA reagent a add 1 volume of BCA reagent B (A: B = 50:1) and
mix well.

##### 2.6.2 Protein content detection

1. Cuvette detection method (sample: BCA working solution = 1:20)
         1) Of 100 each μ L standard and sample to be tested added to
the
reaction tube.
         2) Add 2.0 ml of BCA working solution to each tube and mix.
Incubate at 37 ° C for
30min.
     3) Cool to room temperature. Spectrophotometer detection with a
set wavelength of 562
nm. Correct the instrument
with a cuvette filled with water. Readings were taken within 10 min for all samples.
      4) A standard curve (X-protein concentration UG / ml; y-final od562nm) was
generated by
subtracting the OD value
of the blank well in the standard from the absorbance of the BSA standard (i.e., the final reading). Sample
protein concentrations were calculated according to the standard curve and dilution factor of the samples.

2. Microplate assay method (sample: BCA working solution = 1:8)

   1) G of each 25.0 μ L standard and the sample to be tested
added to the microplate.
      2) 200 μ l was added to each well μ L BCA working solution and
mix thoroughly by shaking for
30s. Close the
microplate and incubate at 37 ° C for 30min.
     3) Cooled to room temperature, the absorbance was detected at the wavelength
range of 540-595 nm on a microplate
reader with 562 nm wavelength as optimal.
      4) A standard curve (X-protein concentration UG / ml; y-final od562nm) was
generated by subtracting the OD value
of the blank well in the standard from the absorbance of the BSA standard (i.e., the final reading). Sample
protein concentrations were calculated according to the standard curve and dilution factor of the samples.

#### 2.7 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/4112/wiki/project/experiments/experiments-pic-2.png)

Fig7.Folding graph of the remaining content of nitrogen and phosphorus elements

#### 2.8 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.

#### 2.9 Fatty acid composition analysis

For fat analysis, total fat was extracted from groups of three independent biological replicates with reference
to the method of Lepage and Roy 1984.

Fatty acids were applied at 30 μ M × 0.25 mm × .The determination was performed by GC-MS of a 0.25 um DB-5
quartz capillary column. The thin tube column was held at 60 ° C for 1 min, then ramped to 160 ° C at a rate of
10 ° C Min 'and raised again to a final 250 ° C at a rate of 2.5 ° C min 1. The injection temperature was 280 °
C, and the injection volume was 1 UL. The mass spectrometry transfer line temperature was 200 ° C, and fatty
acids were identified with an NBS equipped spectral library (National Bureau of Standards, Gaithersburg, MD,
USA) and quantitatively analyzed by determining the comprehensive peak area (Yang et al. 2013, 2014). Finally,
the relative amounts of the detected fatty acids were calculated by normalization.

#### 2.10 Visualization of microalgal oilsomes

1. Divide one sample into three tubes, each filled with 100 μ L stock solution
     2. Add 790 μ L PBS buffer
     3. Add 100 μ Dmso, close nozzle and start shaking, count for 20 min
     4. Open one tube at a time, add 10 μ L Nile red and shaken every 2-3 min
until the end
of 20
min.
     5. Measure at 590 nm wavelength

#### 2.11 Determination of nitrogen and phosphorus in water samples incubated with microalgae

##### 2.11.1 Determination of nitrogen content

1. Digestion: Take a certain amount of the alga like centrifugation. Pipette 400 μl of the supernatant after
centrifugation into 10ml colorimetric tube. Then add 4.6 ml of ultrapure water and 5 ml of digestion agent to
the colorimetric tube. Cover the colorimetric tube and shake it well, tie the lid tightly with tape, put it in a
high pressure steam sterilizer, digest it under high pressure at 120 ℃ for 30 minutes, and take it out after
cooling. Aspirate the clear liquid of the digestive juice for the determination of NO3--N.

In addition, 0, 1, 2, 3, 4, 5, and 6 ml of nitrogen mixed standard solution of 10 μg/ml were respectively drawn
into 7 digestion containers, that is, the nitrogen content in each tube was 0, 10, 20, 30, 40, 50 , 60 μg, to
form a standard series. Then digest simultaneously with the sample according to the above method.

2. Determination of NO3-—N: Since the final pH of the digestate is 2, it exactly meets the pH requirements of
the UV assay, so the supernatant of the digestate can be directly measured at wavelengths of 220 nm and 276 nm
using a 1 cm quartz cuvette. The difference in absorption (A220-2A270) corresponds to the working curve to find
out the corresponding micrograms of nitrogen (μg), divided by the water sample (mg) to get the total nitrogen
content (μg/mg), which is reduced by 10 times to obtain total N %.

##### 2.11.2 Determination of phosphorus content

1. Digestion: Take a certain amount of the alga like centrifugation. Pipette 160 μl of the supernatant after
centrifugation into 10ml colorimetric tube. Then add 1.84 ml of ultrapure water and 2 ml of digestion agent to
the colorimetric tube. Cover the colorimetric tube and shake it well, tie the lid tightly with tape, put it in a
high pressure steam sterilizer, digest it under high pressure at 120 ℃ for 30 minutes, and take it out after
cooling. Aspirate the clear liquid of the digestive juice for the determination of PO 4 3-
—P.

In addition, 0, 1, 2, 3, 4, 5, and 6 ml of phosphorus mixed standard solution of 10 μg/ml were respectively
drawn into 7 digestion containers, that is, the phosphorus content in each tube was 0, 10, 20, 30, 40, 50 , 60
μg, to form a standard series. Then digest simultaneously with the sample according to the above method.

2. Determination of PO43-—P: Add 1ml molybdenum antimony anti-mixing developer and 5ml ultrapure water into the
colorimetric tube and shake well. After 30 minutes of color development at a temperature of 20-40 ℃, at 700 nm,
use an enzyme plate to measure phosphorus on a microplate reader. The measured value A700 of the sample
corresponds to the corresponding phosphorus content (μg) found in the working curve, and divided by the water
sample (mg) to obtain the total phosphorus content (μg/mg), which is reduced by 10 times to obtain the total P%.

### 3 Construction of engineered algal strains

#### 3.1 Microalgal electroporation transformation

1. Prepare Phaeodactylum tricornutum growing to the exponential stage of 1.0E+07~1.0E+08. Sorbitol (375 mm / L)
(i.e. 34.1568 g / 500 ml) RO water configuration, autoclaved. Salmon DNA, heated at 100 ℃ for 1 min. The
electric shock cup is ultrasonically cleaned with RO water, soaked in absolute ethanol, and pre-cooled at -20 ℃.
Filter paper and tweezers are autoclaved.

2. Pour the algal liquid into a 50 ml centrifuge tube on the ultra-clean workbench, 4400 rpm at room
temperature for 5 minutes, take it back to the ultra-clean, pour off the supernatant, and repeat several times
until all the algal liquid is collected. Add 50 ml of sorbitol, suspend with slight shaking, centrifuge at 4400
rpm, discard the supernatant, and repeat three times.

3. According to the actual amount, add an appropriate volume of sorbitol and suspend.

4. To a sterile EP tube (1.5 ml), add 4 μg of linearized plasmid, 4-6 μl (1 μg/μl) of salmon sperm DNA heated
at 100°C for 1 min, and 200 μl of the above-mentioned algal solution. Ice bath for 10min.

5. Use tweezers to remove the electric shock cup from the absolute ethanol, place it on the sterilized filter
paper, and air-dry until the absolute ethanol is completely evaporated.

6. Add the above solution to the shock cup.

7. During electroporation, the metal contacts of the electric shock cup should be dry and wiped clean with
paper. Electroporator parameters: voltage (U): 1500; Capacitance (uF): 25; resistance (Ω): 400; Cuvette (mm): 4.

8. At the end of the electric shock, Pour the algal liquid into a 50 ml conical flask, add 10 ml f/2 seawater
(protect from light). Wrap the conical flask tightly with tin foil and place it in the incubator, dark for 24
hours, and then light again for 24 hours.

9. Coating: Take 5 ml of algae liquid and centrifuge, discard the supernatant, leave a small amount of liquid,
suck it up with a pipette, and evenly point it to the f/2 agarose medium with 85 mg/L bleomycin.

#### 3.2 Validation

##### 3.2.1 Gene level validation

###### 3.2.1.1Total DNA extraction

MolPure ® Plant DNA Kit(18800ES50) adopts MolPure ® DNA Column P1 and a new solution system are suitable for
rapid and simple extraction of genomic DNA from plant tissues (including plants containing phenols,
polysaccharides and enzyme inhibitors), cells and fungal samples.The specific experimental procedures were as
follows.

 1. Sampling: grind 20mg dry weight tissue in liquid nitrogen environment
    2. Splitting: add 400ul LB buffer P1 and 4ul RNase A
    3. Extraction: add 130ul BD BufferP1
    4. Transfer: suck the supernatant into a new centrifuge tube
    5. Settlement: add 1.5 times the volume of PL Buffer P1
    6. Adsorption: transfer to the adsorption column and centrifuge
    7. Washing: add 600ul wash buffer and centrifuge
    8. Residue removal: empty column centrifugation
    9. Elution: add 100ul Elution Buffer, centrifuge
    10. Add water

###### 3.2.1.2Total RNA extraction

TRIeasyTM Total RNA Extraction Reagent(10606ES60) is a total RNA extraction reagent suitable for various
animals and plants, bacterial tissues and cells, with strong lysis ability, which can lyse cells and tissue
samples in a short period of time and effectively inhibit the degradation of RNA in the sample.The specific
experimental procedures were as follows.

###### 1. Sample processing

###### 2.Total RNA extraction

   1) Add 1/5 volume of chloroform to the above lysate
      2) Centrifuge at 4 °C, 12000 g for 10-15 min.
      3) Carefully pipette the upper aqueous phase into a new centrifuge tube and add
1/2 volume of isopropanol
      4) Centrifuge at 4 °C, 12,000 g for 10 min.
      5) Carefully discard the supernatant and add an equal volume of 75% ethanol
Vortex to wash well and flick the bottom of the tube to allow the pellet to levitate.
      6) Centrifuge at 4 °C, 7500 g for 5 min, discard the supernatant, take care not
to lose the RNA pellet.
      7) Place air dry at room temperature for 5-10 min. Add 30-100 μL of RNase-free
water to dissolve RNA

###### 3. Product detection

   1) RNA integrity testing
      2) RNA purity assay

##### 3.2.2 Protein level validation——Western Blot

###### A. Protein sample preparation

   1. Extraction of total protein from monolayer adherent cells
      2. Extraction of total protein from tissues
      3. Extraction of total protein from adherent cells treated with drugs

###### B. Determination of protein content

   1. Extraction of total protein from monolayer adherent cells
      2. Extraction of total protein from tissues
      3. Extraction of total protein from adherent cells treated with drugs

###### C. SDS-PAGE electrophoresis

   1. Wash glass plates
      2. Sizing and loading
      3. Electrophoresis

###### D. Transmembrane

   1. Place the clips used for the transfer, two sponge pads, a glass rod, filter paper, and the
soaked membrane in the enamel dish with transfer fluid.
      2. Open the clamp so that the black side remains horizontal. Pad a sponge pad on
top
      3. Glue the glass plate first by prying it out
      4. Place the clamp in the transfer tank and transfer it with 60 V for 2 h
      5. After turning, the membrane is washed with 1 × Ponceau dye solution was
stained for 5 min (shaking on a destaining shaker). The proteins on the membrane were then visualized by rinsing
away the dye free from the stain with water. Leave the membrane to dry for later use.

###### E. Immune responses

###### F. Chemiluminescence, development, fixation

###### G. Gel image analysis

### 4 Algal sphere

1. Take a certain volume of algal cell concentrate and pre-sterilized SA (sodium alginate) solution 1:1 and mix
well to form the mixture. Shaker 180rpm 21℃ 30min

2. Inject it into a 1ml syringe and drop the mixture at a distance of 20cm from the surface of the pre-cooled
cacl2 solution to form a 4mm diameter algal sphere, fix it at room temperature for 2h, then rinse it with
sterile water three times and place it in seawater spiked with mother liquor for cultivation.

{% 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

#### Overview

Due to COVID-19, our time in the laboratory has been reduced to some extent. As of October 2022, we have
completed most of our experiments. We explored the feasibility of effluent culture of *Phaeodactylum
tricornutum*.
And two transgenic algal strains were successfully constructed, and we found that one of the transgenic strains
(Pha::CA-PRK-eGFP) had significantly higher lipid content and removal rate of nitrogen and phosphorus
elements than the wild type. At the same time, our analysis revealed that the expression levels of genes related
to phosphorus transport and lipid metabolism were significantly up-regulated.

*CA*,*CA2*,and *PRK* genes were obtained by nested PCR cloning using cDNA from *Phaeodactylum
tricornutum* as template. After that, these three genes were successfully recombined into the complex
plasmid vectors *CA-PRK* and *CA2-PRK* to construct the overexpression system. The overexpression
system was successfully transferred into the chassis organism for partial functional validation.

#### Feasibility of wastewater culture of **Phaeodactylum tricornutum**

We configured the effluent medium in a 1:1 volume ratio of f/2 medium to artificial effluent and cultured
wild-type *Phaeodactylum tricornutum*. The test results were as follows.
Cell density: Based on the variation of *Phaeodactylum tricornutum* density, we found that there was no significant
difference between the biomass of *Phaeodactylum tricornutum* in seawater culture and waste water culture.

![](https://static.igem.wiki/teams/4112/wiki/lab/result/fig1-cell-density-line-graph.png)

Fig1. Cell density

It indicates that the waste water culture of *Phaeodactylum tricornutum* is feasible.

#### Obtaining genes

*CA*,CA2 and *PRK* genes were obtained by nested PCR cloning using cDNA of *Phaeodactylum tricornutum* as template. And *CA* and *CA2* were linked to PRK respectively by linker.

Carbonic anhydrase (*CA*) plays a key role in CCM and can reversibly catalyze the CO2 hydration
reaction.
Phosphoribosyl ketokinase (*PRK*) catalyzes the last step of the Calvin cycle reaction and is important for
regulating the flow of sugar in the Calvin cycle.

![](https://static.igem.wiki/teams/4112/wiki/lab/result/fig2-electrophoresis-image-of-the-target-gene.png)

Fig2. Electrophoresis image of the target gene

#### Ligation

The vector we use is pPink-HC-FHG-cp. pPink-HC-FHG-cp is 8678bp in length, contains Paox1 promoter and CYC1
terminator.We use T4 ligase to connect *CA-PRK* and *CA2-PRK* to the vector.

![](https://static.igem.wiki/teams/4112/wiki/lab/part1art.jpg)

Fig3. Overexpression system

#### Transform

The ligated plasmid is transferred to competent cells and cultured. The following is the growth on the medium
containing ampicillin.

![](https://static.igem.wiki/teams/4112/wiki/lab/result/fig4-a-ca-prk-overexpression-system-b-ca2-prk-overexpression-system.png)

Fig4. A CA-PRK overexpression system. B CA2-PRK overexpression system.

#### Positive colony PCR

Pick a single positive colony for culture, and perform PCR detection, and then send the colony with the correct
electrophoresis band to the company for sequencing.

![](https://static.igem.wiki/teams/4112/wiki/lab/engineering/fig-5-colony-pcr-validation-of-agarose-gel-electrophoresis-results.png)

Fig5. Colony PCR validation of agarose gel electrophoresis results

#### Get the plasmid

According to the sequencing results, the bacterial solution was expanded and cultured and the plasmid was
extracted, and the plasmid was verified by PCR detection. Before electroporation transformation, we use the
recombinant plasmid as a template for PCR detection to verify whether our recombinant plasmid contains the
target gene.

#### Electric shock conversion and functional verification

The plasmid was transferred into Pichia pastoris and **Phaeodactylum tricornutum** using an
electroporator.

After screening and culturing, the genomic DNA of **Phaeodactylum tricornutum** was extracted and PCR
was
performed to determine whether the recombinant plasmid was successfully transferred into the algal cells.
Functional validation of transgenic algae strains.

![](https://static.igem.wiki/teams/4112/wiki/lab/result/fig-6-dna-pcr-validation-agarose-gel-electrophoresis-results.png)

Fig6. DNA pcr validation agarose gel electrophoresis results

The protein was extracted and the function of the algal strain was verified at the protein level. The results
showed that our constructed *CA-PRK* overexpression system and CA2-PRK overexpression system have been
successfully introduced into *Phaeodactylum tricornutum*.

![](https://static.igem.wiki/teams/4112/wiki/lab/result/fig-7-coomassie-bright-blue-stain.png)

Fig7. Coomassie bright blue stain

![](https://static.igem.wiki/teams/4112/wiki/lab/result/fig-8-western-blot-verify.jpg)

Fig8. Western Blot verify

We also examined the expression of the target genes, and all of them showed a significant increase, which also
indicates that we successfully transferred the overexpression system into the algal strain

![](https://static.igem.wiki/teams/4112/wiki/lab/result/fig9-histogram-of-gene-level-expression-of-target-genes.png)

Fig9. Histogram of gene level expression of target genes

In batch culture, we examined numerous physiological and biochemical parameters such as density, N and P
content, lipid content, photosynthetic rate, etc. of wild-type *Phaeodactylum tricornutum* and *Phaeodactylum tricornutum*
with *CA-PRK* overexpression system.

It was found that the growth of the transgenic strain was not affected and the biomass was not significantly
different from that of the wild-type strain.

![](https://static.igem.wiki/teams/4112/wiki/lab/result/fig10-cell-density-line-graph.png)

Fig10. Cell density line graph

We stained the treatment with Nile Red fluorescent dye and observed it under LSM. The results are as follows.
The red area represents chlorophyll and the green area represents lipid. It can be seen that the lipid content
of *Phaeodactylum tricornutum* containing *CA-PRK* overexpression system is significantly higher than that of wild
type.

![](https://static.igem.wiki/teams/4112/wiki/lab/result/fig11-transgenic-microalgae-screened-for-antibiotics.png)

Fig11. Transgenic microalgae screened for antibiotics

Quantitative analysis revealed that its lipid content was significantly increased, about 2.3 times higher than
that of the wild type, and correspondingly its protein content and polysaccharide content were reduced. The
increase and decrease in the content of the three metabolites is in line with the widely accepted pattern.

![](https://static.igem.wiki/teams/4112/wiki/lab/result/fig12-histogram-of-lipids-to-dry-weight-content.png)

Fig12. Histogram of lipids to dry weight content

![](https://static.igem.wiki/teams/4112/wiki/project/proof-of-concept/9.png)

Fig13. Glucose content standard curve and Carbonhydrate content histogram

![](https://static.igem.wiki/teams/4112/wiki/practice/education/14.png)

Fig14. Bsa standard curve and protein content histogram

The content of polyunsaturated fatty acids was found to be significantly higher by GC-MS analysis. The content
of ARA and EPA were twice as high as that of wild type. DHA content also has some improvement. This makes sense
for cost reduction of high value products.

![](https://static.igem.wiki/teams/4112/wiki/project/proof-of-concept/11.png)

Fig15. Histogram of the content of the three polyunsaturated fatty acids

#### Gene expression level validation

We found by qpcr that the expression levels of phosphorus transport as well as lipid synthesis-related genes
were significantly higher in *Phaeodactylum tricornutum* containing the *CA-PRK* overexpression system than in the
wild type.

![](https://static.igem.wiki/teams/4112/wiki/lab/result/fig16-qpcr-analysis-of-key-genes.png)

Fig16. qPCR analysis of key genes

![](https://static.igem.wiki/teams/4112/wiki/project/plant/th-fig9-lcm-observation-chart.png)

Fig17. LCM observation Chart

#### Wastewater culture

The growth of the transgenic algae strain did not differ significantly from the wild-type algae strain in the
course of the effluent culture.

![](https://static.igem.wiki/teams/4112/wiki/lab/result/fig18-cell-density-line-graph.png)

Fig18. Cell density line graph

Meanwhile, according to the results of Nile red staining, it can be seen that the lipid accumulation of the
transgenic algae strain is significantly higher than that of the wild-type algae strain.

![](https://static.igem.wiki/teams/4112/wiki/lab/result/fig19-nile-red-fluorescence-content-line-graph.png)

Fig19. Nile red fluorescence content line graph

Overall, our transgenic strain was functionally superior to the wild-type strain.

#### Immobilized Microalgae Beads

We choose immobilized cell technology. Our experimental materials - **Phaeodactylum tricornutum**
were made
into algal spheres using sodium alginate as the embedding material. At the same time, material exchange can
still be carried out between the inside and outside of the algal sphere, which will not affect the normal growth
of **Phaeodactylum tricornutum**.

![](https://static.igem.wiki/teams/4112/wiki/lab/result/fig-20-immobilized-microalgae-actual-photo.png)

Fig20. Immobilized microalgae actual photo

（The numbers marked in the graph are the
number of days of incubation of algal blooms）

It functions well in preventing the escape of algal blooms. No algal plants were observed in the medium at 21
days of incubation.

![](https://static.igem.wiki/teams/4112/wiki/lab/result/fig21-culture-medium-observation-chart.png)

Fig21. Culture medium observation chart

The use of algal spheres together with our designed photobioreactors can further prevent the escape of algal
strains.

#### Cautions

During the whole experiment, aseptic operation should be paid special attention. In past experiments, we have
had a number of experiments that have failed due to contaminated bacteria. This left us a deep impression.

#### Outlook

For the current transgenic algae strain, we are not too comprehensive in testing its physiological and
biochemical indicators, and it still has a lot of places worth exploring. In our future work, we will also
continue to study its various properties and try our best to make it play more and better roles. Of course, in
terms of biosafety, we will also continue to improve the improvement, and strive to design the transfer suicide
switch as soon as possible, so that the transgenic algae strains are more safe and controllable.

{% endblock %}