iGEM 2021 — Team CHINA-FAFU

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# Team:CHINA-FAFU/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

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

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://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)

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# Team:CHINA-FAFU/Engineering

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Team

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[Collaborations](https://2021.igem.org/Team%3ACHINA-FAFU/Collaborations)

Project

[Proof](https://2021.igem.org/Team%3ACHINA-FAFU/Proof_Of_Concept)
[Description](https://2021.igem.org/Team%3ACHINA-FAFU/Description)
[Contribution](https://2021.igem.org/Team%3ACHINA-FAFU/Contribution)
[Sustainability](https://2021.igem.org/Team%3ACHINA-FAFU/Sustainability)
[Implementation](https://2021.igem.org/Team%3ACHINA-FAFU/Implementation)

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LAB

[Design](https://2021.igem.org/Team%3ACHINA-FAFU/Design)
[Engineering](https://2021.igem.org/Team%3ACHINA-FAFU/Engineering)
[Experiments](https://2021.igem.org/Team%3ACHINA-FAFU/Experiments)
[Results](https://2021.igem.org/Team%3ACHINA-FAFU/Results)
[Safety](https://2021.igem.org/Team%3ACHINA-FAFU/Safety)

Model

[Model](https://2021.igem.org/Team%3ACHINA-FAFU/Model)
[Questionnaire Analysis](https://2021.igem.org/Team%3ACHINA-FAFU/Questionnaire)

Practices

[Human\_Practices](https://2021.igem.org/Team%3ACHINA-FAFU/Human_Practices)
[Integrated Human Practice](https://2021.igem.org/Team%3ACHINA-FAFU/Human_Practices/IHP)
[Education](https://2021.igem.org/Team%3ACHINA-FAFU/Education)
[Entrepreneurship](https://2021.igem.org/Team%3ACHINA-FAFU/Entrepreneurship)
[Science Communication](https://2021.igem.org/Team%3ACHINA-FAFU/Science_Communication)
[Education&
Communication](https://2021.igem.org/Team%3ACHINA-FAFU/Communication)

Awards

[Awards](https://2021.igem.org/Team%3ACHINA-FAFU/Awards)

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## Navigation menu

[* 1Overview](#SCIII1)
[* 2Design cycle 1](#SCIII2)
[* 3Design cycle 2](#SCIII3)
[* 4Design cycle 3](#SCIII4)

### Overview

Throughout the project works, we investigated the effect of exogenous addition of AsA on the growth and lipid accumulation of *Phaeodactylum tricornutum.* qPCR detected significant changes in the expression of APX1, DHAR, UGP and VTC2 genes. We successfully cloned VTC2 gene by Nested PCR and synthesized APX1, DHAR and UGP genes. After that, these four genes were successfully recombined into the plasmid vector to construct the overexpression system. After that, the overexpression system was transferred to Pichia pastoris for functional validation. Finally, the results of the transformation were tested and validated in *Phaeodactylum tricornutum.* to investigate whether the overexpression system was successfully expressed in *Phaeodactylum tricornutum.* and its effect on its growth and metabolism, as well as whether its antioxidant stress capacity, lipid content and fucoxanthin content were increased.

There are three engineering iterations in our project. First, in the first cycle, we treat the selected algal species under specific conditions and screen for optimal treatment conditions and target genes. Secondly, in the second cycle, we further define the treatment conditions and perform expression vector construction and expression validation. Finally, in the third cycle, we transferred the vectors into *Pichia pastoris* and *Phaeodactylum tricornutum* for further functional validation.

---

# Design Cycle 1: Optimal treatment concentration and target gene screening of AsA in *Phaeodactylum tricornutum*

The principle of our engineered algal strain is to achieve antioxidant protection of
its cellular chloroplasts from oxidative damage by stimulating the production of
endogenous ascorbic acid. ROS act as intracellular signaling molecules regulating
plant development, programmed cell death, and response to biotic and abiotic
stresses. AsA is directly involved in the scavenging of ROS, and the AsA-GSH cycle
plays a very important role in the scavenging of ROS.
`
At the beginning of the project, we wanted to investigate the effect of high
concentrations of ascorbic acid on microalgae and, at the same time, to verify the
soundness of the project idea. We treated microalgae with different concentrations
of ascorbic acid and measured their physiological and biochemical parameters to
reflect the effect of AsA on microalgae cells. (For more details, visit Design)

![](https://static.igem.org/mediawiki/2021/d/da/T--CHINA-FAFU--eng2.png)

##### Fig.1 AsA synthesis pathway and ROS clearance mechanism in plants

![](https://static.igem.org/mediawiki/2021/3/3b/T--CHINA-FAFU--gctj1.png)

##### Table 1 Measurement indicators and tools used

![](https://static.igem.org/mediawiki/2021/0/01/T--CHINA-FAFU--gctj2.png)

##### Fig.2 qPCR analysis of APX1, UGP, VTC2, DHAR gene expression in the VC-250 treatment group

In the first engineering iteration, we found that the addition of certain
concentrations of exogenous AsA promoted the photosynthetic efficiency of cell
chloroplasts and led to an increase in cell lipids, unsaturated fatty acids, and
fucoxanthin content. This led us to great interest. We performed qPCR on the genes
of lipid synthesis pathway, rockweed flavin metabolism pathway, ascorbic acid
metabolism pathway, and N/P metabolism pathway of the treated microalgae and finally
screened four genes that met our expectation for the next engineering operation.

---

# Design Cycle 2: Gene acquisition and expression vector construction

#### Get gene

Using the cDNA obtained by reverse transcription of *Phaeodactylum tricornutum* RNA as
a template, the VTC2 gene was cloned by Nested PCR. These three genes were
synthesized according to the APX1, DHAR and UGP gene sequences obtained from NCBI.

![](https://static.igem.org/mediawiki/2021/6/65/T--CHINA-FAFU--gctj3.png)

##### Fig.3 Principle of Nested PCR

Nested PCR has a very high specificity, which can guarantee the accuracy of the
genes cloned by our PCR.

![](https://static.igem.org/mediawiki/2021/7/74/T--CHINA-FAFU--gctj4.png)

##### Fig.4 Electrophoresis image of the target gene

APX1 and DHAR are the key genes in the AsA-GSH cycle and participate in the
regeneration of AsA. VTC2 encodes GGPase, which catalyzes the reaction between
GDP-L-galactose and GTP to produce L-galactose-1-phosphate. Studies have proved that
VTC2 is the seventh rate-limiting gene in the process of ascorbic acid synthesis.
UGPase encoded by UGP catalyzes UTP reaction to produce UDPG, which is an important
substance in the synthesis and metabolism of plant organisms.

---

### Ligation

The vector we use is pPink-HC-FHG-cp. pPink-HC-FHG-cp is 8678bp in length, contains
Paox1 promoter and CYC1 terminator, and is pre-linked with sfGFP gene.We use T4
ligase to connect APX1, DHAR, UGP and VTC2 to the vector.

![](https://static.igem.org/mediawiki/2021/7/75/T--CHINA-FAFU--gongcheng2.png)

##### Fig.5 pPink-HC-FHG-cp plasmid map

Overexpression system: We inserted APX1, DHAR, UGP, and VTC2 between the promoter
and sfGFP, respectively.

![](https://static.igem.org/mediawiki/2021/6/6d/T--CHINA-FAFU--gctj5.png)

##### Fig.6 Overexpression system

A: APX1 overexpression system. B: DHAR overexpression system. C: UGP
overexpression system. D: VTC2 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.org/mediawiki/2021/8/8c/T--CHINA-FAFU--gongchenmg3.png)

##### Fig.7 A: APX1 overexpression system. B: DHAR overexpression system. C: UGP overexpression system. D: VTC2 overexpression system

---

### Positive colony PCR

Potentially positive single colonies were picked for incubation and subjected to
PCR, followed by electrophoresis of the correct bands and sequencing.

![](https://static.igem.org/mediawiki/2021/9/91/T--CHINA-FAFU--gctj6.png)

##### Fig.8 VTC2. B:UGP. C:DHAR. D:APX1

##### A: VTC2 overexpression system. B: UGP overexpression system. C: DHAR overexpression system. D: APX1 overexpression system.

---

### 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. The results are as follows, the recombinant plasmid contains the target
gene.

![](https://static.igem.org/mediawiki/2021/9/9a/T--CHINA-FAFU--GGGGC2.png)

##### Fig.9 Plasmid PCR results

---

# Design Cycle 3: Obtain engineered yeast and algae strains

### Electric shock conversion and functional verification

1.Use an electroporator to transfer the plasmid into *Pichia pastoris* and
*Phaeodactylum tricornutum*

2.Use the Yeast Genomic DNA Extraction Kit to extract the genomic DNA of *Pichia pastoris* , and use the CTAB method to extract the DNA of  *Phaeodactylum tricornutum*.

3.Use the extracted DNA of the chassis organism as a template for PCR detection, and
judge whether the recombinant plasmid is introduced into the chassis organism
according
to the PCR results.

![](https://static.igem.org/mediawiki/2021/d/d7/T--CHINA-FAFU--GGGGC1.png)

##### Fig.10 PCR detection of DNA

After electroporation transformation, we expanded *Phaeodactylum tricornutum* and then
extracted its DNA using the CTAB method, and used the DNA as a template to detect
whether the recombinant plasmid gene was introduced into the chassis organism by
PCR. The results showed that the VTC2 overexpression system and UGP overexpression
system we constructed were successfully introduced into *Phaeodactylum tricornutum*.

4.Use the Total Protein Extraction Kit to extract the proteins of the chassis
organisms,
and use the Enzyme Activity Detection Kit to detect the activity of DHAR, APX, GGP,
UGP,
and explore whether the enzyme activity of the chassis organisms that have been
successfully introduced into the recombinant plasmid has increased.

5.Detect the expression of genes related to AsA synthesis and regeneration pathways,
fucoxanthin synthesis and metabolism pathways by qPCR, and explore the influence of
the
successful introduction of recombinant plasmids on the molecular mechanism of
chassis
organism AsA synthesis and regeneration.

6. Use microplate reader, LSM, GC-MS to detect the lipid content and components of
the
successfully transformed *Phaeodactylum tricornutum*. Use a microplate reader to
detect
its fucoxanthin content.

![](https://static.igem.org/mediawiki/2021/a/a7/T--CHINA-FAFU--gongcheng22haotianjia.jpeg)

##### Fig.11 LSM photographs of *Phaeodactylum tricornutum* containing an overexpression system stained with BODIPY 505/515.Images were acquired randomly from at least 20 cells per sample, and typical images are presented here. Bar was shown in the fifigure.

We added bleomycin to screen  *Phaeodactylum tricornutum* containing UGP overexpression
system and VTC2 overexpression system. The screened  *Phaeodactylum tricornutum* was
dyed with BODIPY 505/515 fluorescent dye and observed under a laser confocal
microscope.
The excitation wavelength is 488nm and the emission wavelength is 530nm. The results
are as follows. The red area represents chlorophyll and the green area represents
lipids.
The total oil content of  *Phaeodactylum tricornutum*  in the VTC2
overexpression system
was significantly higher than that of the wild type. The chlorophyll content of
 *Phaeodactylum tricornutum*  in the UGP overexpression system was
significantly higher
than that of the wild type.

---

### Function prediction

Studies have found that the levels of VTC2 and AsA in Chlamydomonas reinhardtii are
significantly increased during oxidative stress. VTC2 is a highly regulated enzyme
involved in the synthesis of AsA in Chlorella. It works with the ascorbic acid
circulation system to help cells resist oxidative stress. [1]. VTC2 and
VTC5 in
Arabidopsis are involved in the synthesis of GGP and are regulated by light
[2].
Therefore, we speculate that if the composite part [BBa\_K3812010](https://2021.igem.org/Team%3ACHINA-FAFU/Parts) is
successfully expressed in
the
chassis organisms, the activity of GGP and the AsA content in the chassis organisms
will
be significantly increased, and the ability of the chassis organisms to eliminate
ROS
and resist oxidative stress will also be significantly improved.
It has been studied that after overexpression of DHAR in the cytoplasm of
Arabidopsis in
tobacco, the AsA content in tobacco increased by nearly 2 times [3].
After
expressing
DHAR in wheat cytoplasm in tobacco and corn, the AsA content of tobacco and corn
leaves
increased by 4 times, the DHA content decreased, and the AsA/DHA ratio increased
[4].
Therefore, we speculate that the AsA content in chassis organisms containing
composite
parts [BBa\_K3812008](https://2021.igem.org/Team%3ACHINA-FAFU/Parts) will
increase significantly, the ratio of AsA/DHA may also increase, and
the
regeneration rate of AsA may be significantly accelerated.
Both APX1 and DHAR participate in the recycling process of AsA, and APX1 mainly
promotes
the degradation of H2O2 to H2O, eliminating the
damage of H2O2 to intracellular
organelles such as chloroplasts and mitochondria. Therefore, we speculate that the
AsA-GSH cycle in the chassis organisms containing the composite part [BBa\_K3812007](https://2021.igem.org/Team%3ACHINA-FAFU/Parts) will be
accelerated, and the ability to remove ROS will be significantly increased. It is
more
conducive to cell resistance to oxidative stress, maintaining the stability of the
intracellular membrane structure and organelle structure and the improvement of
functions.
UGP is mainly involved in sucrose synthesis, cell wall formation, and response to
abiotic stress in higher plants [5-8]. The purpose of our construction of
UGP
overexpression system [BBa\_K3812009](https://2021.igem.org/Team%3ACHINA-FAFU/Parts) is to
promote the synthesis of organic matter and
accelerate
the growth of chassis biological cells.

---

### References:

[1]Urzi Ca E I, Adler L N, MD Page, et al. Impact of Oxidative Stress on Ascorbate
Biosynthesis in Chlamydomonas via Regulation of the VTC2 Gene Encoding a
GDP-l-galactose
Phosphorylase[J]. Journal of Biological Chemistry, 2012, 287(17):14234-45.

 [2]Dowdle J, Ishikawa T, Gatzek S, et al. Two genes in Arabidopsis thaliana
encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and
seedling viability.[J]. Plant Journal for Cell & Molecular Biology, 2010,
52(4):673-689.

 [3]Amin Elsadig, Eltayeb,Naoyoshi, Kawano,Ghaz, et al. Overexpression of
monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to
ozone, salt and polyethylene glycol stresses[J]. Planta, 2007, 225(5):1255-1264.

 [4]Chen Z , Young T E , Ling J , et al. Increasing vitamin C content of plants
through enhanced ascorbate recycling[J]. Proceedings of the National Academy of
Sciences
of the United States of America, 2003, 100(6).

 [5]Winter H, Huber SC. Regulation of sucrose metabolism in higher plants:
localization and regulation of activity of key enzymes[J]. Crit Rev Plant Sci.
2000,19:
31-67.

 [6]Flores-Diaz M. Alape-Giro'n A, Persson B, et al. Cellular UDP-glucose
deficiency
caused by a single point mutation in the UDP-glucose pyrophosphorylase[J]. J Biol
Chem.
1997, 272: 23784-2379.

 [7]Bishop JD, Moon BC, Harrow F, et al. A second UDP-glucosepyrophosphorylase
is required for differentiation and development in Dictyosteliumdiscoideum[J]. J
Biol Chem. 2002, 277: 32430-32437.

 [8]Repetto O, Bestel-Corre G, Dumas-Gaudot E, et al. Targeted proteomics to
identify cadmiuminduced protein modifications in Glomusmosseae-inoculated pea
roots[J]. New Phytol. 2003, 157: 555-567.

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# Team:CHINA-FAFU/Experiments

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[Proof](https://2021.igem.org/Team%3ACHINA-FAFU/Proof_Of_Concept)
[Description](https://2021.igem.org/Team%3ACHINA-FAFU/Description)
[Contribution](https://2021.igem.org/Team%3ACHINA-FAFU/Contribution)
[Sustainability](https://2021.igem.org/Team%3ACHINA-FAFU/Sustainability)
[Implementation](https://2021.igem.org/Team%3ACHINA-FAFU/Implementation)

[Parts](https://2021.igem.org/Team%3ACHINA-FAFU/Parts)

LAB

[Design](https://2021.igem.org/Team%3ACHINA-FAFU/Design)
[Engineering](https://2021.igem.org/Team%3ACHINA-FAFU/Engineering)
[Experiments](https://2021.igem.org/Team%3ACHINA-FAFU/Experiments)
[Results](https://2021.igem.org/Team%3ACHINA-FAFU/Results)
[Safety](https://2021.igem.org/Team%3ACHINA-FAFU/Safety)

Model

[Model](https://2021.igem.org/Team%3ACHINA-FAFU/Model)
[Questionnaire Analysis](https://2021.igem.org/Team%3ACHINA-FAFU/Questionnaire)

Practices

[Human\_Practices](https://2021.igem.org/Team%3ACHINA-FAFU/Human_Practices)
[Integrated Human Practice](https://2021.igem.org/Team%3ACHINA-FAFU/Human_Practices/IHP)
[Education](https://2021.igem.org/Team%3ACHINA-FAFU/Education)
[Entrepreneurship](https://2021.igem.org/Team%3ACHINA-FAFU/Entrepreneurship)
[Science Communication](https://2021.igem.org/Team%3ACHINA-FAFU/Science_Communication)
[Education&
Communication](https://2021.igem.org/Team%3ACHINA-FAFU/Communication)

Awards

[Awards](https://2021.igem.org/Team%3ACHINA-FAFU/Awards)

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![](../../image/医疗44.png)

## Menu navigation

[* 1General](#General)
[* 2Microalgae](#Microalgae)
[* 3DNA](#DNA)
[* 4RNA](#RNA)
[* 4Yeast](#Yeast)

---

# 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 dd-water.

#### Liquid medium

#### 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

#### Algal species and culture

#### Microalgae cell count and growth curve determination

##### Microalgae 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 .

##### 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.

#### Microalgae culture ascorbic acid concentration setting

Use ultrapure water by autoclaving and deaerating 121°C for 20 min, cool
to room temperature and add different amounts of L-ascorbic acid.

#### 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.

#### 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.

#### Extraction and content determination of total ascorbic acid from microalgae

##### Microalgae Total Ascorbic Acid Extract

Weigh the sample in a mortar, add 50g/L TCA solution, grind it into a
slurry in an ice bath,
transfer it to a 100ml volumetric flask and fix the volume to the scale with 50g/L TCA
solution,
mix and extract for 10min, then filter and collect the filtrate.

##### Ascorbic acid standard curve making

Seven test tubes were taken, numbered, and various solutions were added
according to Table 1,
and the mixture was placed at 30 ℃ for 60 min, and then the absorbance value was measured at
the
wavelength of 534 nm using the mixture of test tube 0 as reference. The regression equation
was
obtained by converging the ascorbic acid mass as the horizontal coordinate and absorbance as
the
vertical coordinate to the standard curve.

![](https://static.igem.org/mediawiki/2021/8/8d/T--CHINA-FAFU--exp11.png)

##### Determination of reduced ascorbic acid content

Take 1ml of sample extract in a test tube, add 1ml of 50g/L TCA
solution, and then react and
measure according to the same method of making standard curve. Record the absorbance of the
reaction system at the wavelength of 534 nm. Repeat 3 times.

##### Determination of total ascorbic acid content

Take 1 ml of the extract from the sample, add 0.5 ml of 60 mmol/L
DTT-acetic acid solution, then
adjust the pH to 7~8 with Na2HPO4-NaOH solution and leave it at room temperature for 10 min
to
reduce the dehydroascorbic acid. Then add 0.5ml of 0.2g/ml trichloroacetic acid solution and
adjust the pH to 1~2. The total ascorbic acid content was calculated by the same method of
ascorbic acid determination, and the original reduced ascorbic acid content in the sample
was
subtracted from it to obtain the dehydroascorbic acid content.

#### Extraction and determination of Fucoxanthin from microalgae

##### 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.org/mediawiki/2021/b/b4/T--CHINA-FAFU--exp12.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. tricornutum*was 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

#### 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.

#### 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).

#### 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)

#### Visualization of microalgal oilsomes

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.

# DNA

#### DNA concentration determination

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

#### DNA electrophoresis

#### DNA purification(TRANSTM)

#### Plasmid DNA extraction(TRANSTM)

#### DNA Ligase(infusion)

#### DNA Ligase(T4)

#### PCR(pfu ultra enzyme)

#### PCR(Taq enzyme)

#### Incision enzyme(TRANSTM)

#### Competent Cell(TRANSTM)

# 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.

# Yeast

#### Yeast receptor cells

1. Transfer the yeast solution of logarithmic growth period to a sterile centrifuge tube and
centrifuge at 4℃ for 1500g for 5min.
2. Add ice-cold sterile water to the centrifuge tube, shake to resuspend the precipitate,
centrifuge at 4°C for 1500g, 5min, and repeat several times.
3. Add a certain amount of 1M sorbitol to the centrifuge tube, resuspend and dispense. This
bacterium is the pPink4 receptor cells.

#### Yeast electroshock conversion

1.Mix the prepared pPink4 receptor cells with a certain amount of linearized plasmid DNA and
transfer to a pre-cooled electroshock cup
2. Place the electroshock cups in an ice bath for 5 min.
3. Perform electroshock using the Pythium mode of the electroshock transformer.
4. Immediately after the electroshock treatment, add ice-cold YPDS medium and mix well and
incubate at 28°C for 2-12 hours.
5. After incubation, the broth is coated on PDA screening medium and incubated at 28°C for
3-10d
until a monoclonal is formed.

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## Navigation menu

[* 1Overview](#SCIII1)
[* 2The effect of AsA on the growth and metabolism of microalgae](#SCIII2)
[* 3The test results are as follows](#SCIII3)
[* 4Get the gene](#SCIII4)
[* 5Ligation](#SCIII5)
[* 6Transform](#SCIII6)
[* 7Positive colony PCR](#SCIII7)
[* 8Get the plasmid](#SCIII8)
[* 9Electric shock conversion and functional verification](#SCIII9)

### Overview

 **Due to the impact of *COVID-19*, our time in the laboratory has been reduced to a certain extent. As of October 2021, we have completed part of the experimental design part. We explored the effect of exogenous AsA on the growth and lipid accumulation of Phaeodactylum tricornutum. We found that when the amount of AsA added to *Phaeodactylum tricornutum* was 250mg/L, its lipid content and growth rate were the highest relative to the control group and other treatment groups, and its fucoxanthin content and the value of Fv/Fm increased significantly. At the same time, we detected by qPCR that APX1, DHAR, UGP and VTC2 gene expression levels were significantly up-regulated.**
Using the cDNA of *Phaeodactylum tricornutum* as a template, we successfully cloned the VTC2 gene by Nested PCR, and successfully synthesized  **the APX1, DHAR and UGP genes in *Ailurus vecTM* company.** Afterwards, these four genes were successfully recombined into a plasmid vector to construct an overexpression system.

 **Now we are trying to transfer the overexpression system to the chassis organism-*Pichia pastoris* for functional verification. Next, we will transfer the expression system to *Phaeodactylum tricornutum*** to continue the detection and verification of the transformation results. We expect to complete the unfinished experimental design and conduct a complete display before 2021 Giant Jamboree.

---

### The effect of AsA on the growth and metabolism of microalgae

We first added different concentrations of AsA to the *Phaeodactylum tricornutum*
culture medium, VC-CK: control group, no AsA added; VC-80: AsA added at 80mg/L;
VC-160: AsA added at 160mg/L; VC-200: the addition amount of AsA is 200mg/L; VC-250:
the addition amount of AsA is 250mg/L; VC-300: the addition amount of AsA is
300mg/L. During the cultivation process, we carried out the following physiological
and biochemical indicators and gene expression tests:

![](https://static.igem.org/mediawiki/2021/3/3b/T--CHINA-FAFU--gctj1.png)

##### Table 1 Measurement indicators and tools used

### The test results are as follows

#### Cell density

According to the changes in the density of *Phaeodactylum tricornutum*, we speculate
that the addition of 250mg/L of AsA may be the most conducive to the growth of
*Phaeodactylum tricornutum* .

![](https://static.igem.org/mediawiki/2021/a/ad/T--CHINA-FAFU--RES22.png)

##### Fig.1 Density of *Phaeodactylum tricornutum* under different concentrations of AsA

**#### Chlorophyll fluorescence parameters**

![](https://static.igem.org/mediawiki/2021/2/25/T--CHINA-FAFU--re_2.jpg)

##### Fig.2 Fv/Fm values of *Phaeodactylum tricornutum* under different concentrations of AsA.Asterisks (\*) indicates significant difference by t test from no AsA treatment ( P < 0.05; Student’s t test)

According to the Fv/Fm value, we judge that 250mg/L of AsA may be the most
beneficial for the photosynthesis of *Phaeodactylum tricornutum*

**#### Fucoxanthin content**
We extracted the *Phaeodactylum tricornutum* fucoxanthin in the VC-250
treatment group and tested the content with a microplate reader. The
results showed that the content of *Phaeodactylum tricornutum*
fucoxanthin in the VC-250 treatment group was higher than that of
the control group. About 1.5 times.

![](https://static.igem.org/mediawiki/2021/0/07/T--CHINA-FAFU--rs4.png)

##### Fig.3 Fucoxanthin content of *Phaeodactylum tricornutum* in the control group and the VC-250 treatment group.Asterisks (\*) indicates significant difference by t test from no AsA treatment (P < 0.05; Student’s t test)

**#### Lipid content**
According to the changes in the intracellular lipid content of *Phaeodactylum tricornutum*, we judged that the addition of 250mg/L of AsA may be the most conducive
to the accumulation of lipids of *Phaeodactylum tricornutum.*

![](https://static.igem.org/mediawiki/2021/7/76/T--CHINA-FAFU--re_3.jpg)

##### Fig.4 Lipid content of *Phaeodactylum tricornutum* under different concentrations of AsA Asterisks (\*) indicates significant difference by t test from no AsA treatment after 9 days. (P < 0.05; t test)

![](https://static.igem.org/mediawiki/2021/c/c4/T--CHINA-FAFU--rs3000.png)

##### Fig.5 LSM observation of the fat body of *Phaeodactylum tricornutum* stained with BODIPY dye on the day of AsA culture

![](https://static.igem.org/mediawiki/2021/7/76/T--CHINA-FAFU--rs7.png)

##### Fig.6 The percentage of lipid content of *Phaeodactylum tricornutum* in the control group and the VC-250 treatment group (the percentage of the dry weight of the lipid in the dry cell weight).Asterisks (\*) indicates significant difference by t test from no AsA treatment (P < 0.05; Student’s t test).

According to the changes in the intracellular lipid content
of *Phaeodactylum tricornutum*, we judged that the addition of
250mg/L of AsA may be the most conducive to the accumulation
of lipids of *Phaeodactylum tricornutum*.

#### Lipid components

We used GC-MS to analyze the composition of lipids produced
by *Phaeodactylum tricornutum* in the control group and the
VC-250 treatment group. The results showed that the content
of monounsaturated fatty acids in the VC-250 treatment group
increased by about 9% compared to the control group. The
content is reduced by about 15%, the polyunsaturated fatty
acid content is increased by about 12%, and the total oil
content is increased by about 25%.

![](https://static.igem.org/mediawiki/2021/7/79/T--CHINA-FAFU--re_1.jpg)

##### Table 2 Lipid composition of *Phaeodactylum tricornutum* in VC-250 treatment group(% of total FAs ± SD, n = 3) Three groups were taken for the determination of fatty acid composition.FA, fatty acid; DW, dry weight; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; ND, not detected.

#### Gene expression

The results showed that the expressions of APX1, UGP, VTC2,
and DHAR genes in the VC-250 treatment group were
up-regulated by about 9, 7.5, 3.5, and 7.5 times,
respectively.

![](https://static.igem.org/mediawiki/2021/c/c8/T--CHINA-FAFU--rs8.png)

##### Fig.7 qPCR analysis of APX1, UGP, VTC2, DHAR gene expression in the VC-250 treatment group

---

### Get gene

Using the cDNA obtained by reverse transcription of *Phaeodactylum tricornutum* RNA
as a
template, the VTC2 gene was cloned by Nested PCR. These three genes were synthesized
according to the APX1, DHAR and UGP gene sequences obtained from NCBI.

![](https://static.igem.org/mediawiki/2021/a/a0/T--CHINA-FAFU--eng3.png)

##### Fig.8 Principle of Nested PCR

Nested PCR has a very high specificity, which can guarantee the accuracy of the genes
cloned by our PCR.

![](https://static.igem.org/mediawiki/2021/b/b3/T--CHINA-FAFU--rs10.png
                                    )

##### Fig.9 Electrophoresis image of the target gene

APX1 and DHAR are the key genes in the AsA-GSH cycle and participate in the regeneration
of AsA. VTC2 encodes GGPase, which catalyzes the reaction between GDP-L-galactose and
GTP to produce L-galactose-1-phosphate. Studies have proved that VTC2 is the seventh
rate-limiting gene in the process of ascorbic acid synthesis. UGPase encoded by UGP
catalyzes UTP reaction to produce UDPG, which is an important substance in the synthesis
and metabolism of plant organisms.

---

### Ligation

The vector we use is pPink-HC-FHG-cp. pPink-HC-FHG-cp is 8678bp in length, contains
Paox1 promoter and CYC1 terminator, and is pre-linked with sfGFP gene.We use T4 ligase
to connect APX1, DHAR, UGP and VTC2 to the vector.

![](https://static.igem.org/mediawiki/2021/7/75/T--CHINA-FAFU--gongcheng2.png)

##### Fig.10 pPink-HC-FHG-cp plasmid map.

Overexpression system: We inserted APX1, DHAR, UGP, and VTC2 between the promoter and
sfGFP, respectively.

![](https://static.igem.org/mediawiki/2021/6/6d/T--CHINA-FAFU--gctj5.png)

##### Fig.11 A: APX1 overexpression system. B: DHAR overexpression system. C: UGP overexpression system. D: VCT2 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.org/mediawiki/2021/8/8c/T--CHINA-FAFU--gongchenmg3.png)

##### Fig.12 A: APX1 overexpression system. B: DHAR overexpression system. C: UGP overexpression system. D: VCT2 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.org/mediawiki/2021/9/91/T--CHINA-FAFU--gctj6.png)

##### Fig.13 A:VTC2. B:UGP. C:DHAR. D:APX1.

### 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. The results are as follows, the recombinant plasmid contains the target
gene.

![](https://static.igem.org/mediawiki/2021/9/9a/T--CHINA-FAFU--GGGGC2.png)

##### Fig.14 Plasmid PCR results

---

### Electric shock conversion and functional verification

The plasmid was transferred into *Pichia pastoris* and *Phaeodactylum tricornutum*
using an
electroporator. As of October 2021, the system we have built is still being tested and
verified. Please go to our Engineering section for detailed plans. We will continue to
complete subsequent experiments in accordance with Engineering's plan.Here are some of
the test results we have achieved:

![](https://static.igem.org/mediawiki/2021/d/d7/T--CHINA-FAFU--GGGGC1.png)

##### Fig.15 PCR detection of DNA

After electroporation transformation, we expanded Phaeodactylum tricornutum and then
extracted its DNA using the CTAB method, and used the DNA as a template to detect
whether the recombinant plasmid gene was introduced into the chassis organism by
PCR. The results showed that the VTC2 overexpression system and UGP overexpression
system we constructed were successfully introduced into Phaeodactylum tricornutum.

![](https://static.igem.org/mediawiki/2021/2/23/T--CHINA-FAFU--re_4.jpg)

##### Fig.16 LSM photographs of *Phaeodactylum tricornutum* containing an overexpression system stained with BODIPY.Images were acquired randomly from at least 20 cells per sample, and typical images are presented here. Bar was shown in the fifigure.

We added bleomycin to screen *Phaeodactylum tricornutum* containing UGP overexpression
system and VTC2 overexpression system. After screening, *Phaeodactylum tricornutum* was
dyed with BODIPY fluorescent dye and observed under LSM. The excitation wavelength is
488nm and the emission wavelength is 530nm. The results are as follows. The red area
represents chlorophyll and the green area represents lipids. The total oil content of
*Phaeodactylum tricornutum* in the VTC2 overexpression system was significantly higher
than that of the wild type. The chlorophyll content of *Phaeodactylum tricornutum* in the
UGP overexpression system was significantly higher than that of the wild type.

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