iGEM 2024 — Team china-fuzhou


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1 Iteration One
Determination of Carotenoids

1.1 Design

1.2 Build

1.3 Test

1.4 Learning

![](https://static.igem.wiki/teams/5385/footer/footerline.png)

2 Iteration Two
Determination of Astaxanthin

2.1 Design

2.2 Build

2.3 Test

2.4 Learning

![](https://static.igem.wiki/teams/5385/footer/footerline.png)

3 Iteration Three
Preliminary Bioinformatics Analysis

3.1 Design

3.2 Build

3.3 Test

3.4 Learning

![](https://static.igem.wiki/teams/5385/footer/footerline.png)

4 Iteration Four
Gene Transformation

4.1 Design

4.2 Build

4.3 Test

4.4 Learning

![](https://static.igem.wiki/teams/5385/footer/footerline.png)

References

# 1 Iteration One Determination of Carotenoids

## 1.1 Design

β-Carotene, as a precursor of astaxanthin, is widely found in higher
plants. We plan to construct an expression system for astaxanthin in
longan fruits, and the first step is to extract and detect the
carotenoid content in longan tissues.

According to the carotenoid extraction methods consulted, commonly
employed methods include supercritical fluid extraction, enzymatic
catalysis, and ultrasonic or microwave assisted extraction method based
on organic solvent. After analyzing and comparing these methods, we
found that microwave-assisted technology can improve the extraction
efficiency and yield of astaxanthin but may reduce the biological
activity of the extract. After discussing with our supervisor, we
decided to use a carotenoid detection kit. The extraction principle
involves mixing the sample with an organic solvent to separate
carotenoids from non-carotenoid components. Carotenoids have a
characteristic absorption peak at 440nm. This method has good extraction
efficiency and is relatively simple to operate.

## 1.2 Build

Using the Carotenoid Extraction Kit, Carotenoid Reagent 1 (product
solution formula undisclosed): Liquid, 1000 mL × 2 bottles, stored at
4°C. Reagent 2 (product solution formula undisclosed): Liquid, 1000 mL ×
1 bottle, stored at 4°C.Prior to use, mix Reagent 1 and Reagent 2 in a
ratio of 2:1 to form the Extracting Solution, and store it sealed at 4°C
to prevent evaporation.

## 1.3 Test

Add approximately 6 ml of the Extracting Solution to 0.2 g of tissue and
allow it to soak for 20 minutes. Repeat the extraction process until the
solution becomes colorless. Combine the extracts and adjust the total
volume to 50 ml. Centrifuge the solution at 5000 rpm for 10 minutes, and
then take the supernatant to measure its absorbance at 440 nm using a
spectrophotometer. The final result can be obtained through a
calculation formula.

$$ \text{Carotenoid content } (\mu g/g) = \frac{\Delta A \cdot V
}{(\varepsilon \cdot d) \cdot W} \cdot 1000 = \frac{20 \cdot \Delta
A}{W} $$

V: total volume of extract, 50mL;

ε: Empirical absorbance coefficient of carotenoids, 2500;

d: cuvette aperture, 1cm;

W: Sample mass, g

![Figure 1 Extraction of carotenoids](https://static.igem.wiki/teams/5385/engineering/1.png)

Figure 1 Extraction of carotenoids

## 1.4 Learning

The results showed that longan pulp, leaves, and other parts contain
abundant carotenoids. Among them, the carotenoid content in mature
leaves is higher than that in young leaves, while the content in longan
pericarp is relatively low, and there is almost no carotenoid in the
seed.

![Figure2 Histogram of carotenoid content](https://static.igem.wiki/teams/5385/engineering/2.png)

Figure 1 Extraction of carotenoids

During the measurement process, we noticed that the extraction solution
is volatile and toxic, so protective measures should be taken during
operation. The measurement must be carried out quickly to prevent errors
caused by volatilization.

# 2 Iteration Two Determination of Astaxanthin

## 2.1 Design

Based on the results of Iteration One, we preliminarily believe that our
design has certain theoretical feasibility. Therefore, we further
narrowed the target range of longan tissues, and continued to extract
astaxanthin from tissues with high carotenoid content—flesh and leaves.
Astaxanthin is a type of carotenoid, a liposoluble pigment that is
insoluble in water but soluble in certain organic solvents such as
acetone.

## 2.2 Build

Based on the reviewed literature, we have designed a method for
extracting astaxanthin from longan fruits and leaves. DMSO was used as
the extraction reagent. We will use instruments such as a balance, oven,
100 mesh sieve, glass tube, microplate reader, 96-well plate,50 ml
centrifuge tubesand[2][3][4].

## 2.3 Test

The astaxanthin content is determined using the spectrophotometric
method. Extract 10 mg of tissue powder with 5mLDMSO in a glass tube for
10 min, and then centrifuge the mixture at 4,000 rpm for 10 min to
collect the supernatant.It is important to note that the extraction
process should be performed away from light. Finally, the red
supernatant is diluted to 25 mL with the extraction solvent, and the
absorbance is measured at 530 nm using the Microplate reader.

## 2.4 Learning

The results of extracting astaxanthin from longan leaves and fruits were
not ideal. At the apparent level, we found that the content of
astaxanthin in both longan leaves and fruits is very low. This prompts
us to conduct further exploration at the genetic level.

# 3 Iteration Three Preliminary Bioinformatics Analysis

## 3.1 Design

After reviewing a substantial amount of relevant literature, we have
tentatively identified the
CrBKT gene and the
HpBHY gene as having significant
impacts on astaxanthin synthesis. Our plan is to screen the longan
genome for endogenous genes that may have homologous relationships with
these two genes.

## 3.2 Build

Based on previous research, locate the corresponding gene sequences
(GenBank No. AY860820 and GenBank No. BD250390) and use the online
analysis tool ExPASy Web (Artimo et al., 2012) (<https://web.expasy.org/translate/>)to translate them into amino acid sequences[1]. Utilize the TBtools
analysis tool to conduct a preliminary BLAST comparison of the
translated amino acid sequences with the longan genome. Compare the
suspected homologous genes with the amino acid sequences of
CrBKT and
HpBHY genes to construct a
phylogenetic tree in MEGA11.0 software using the maximum likelihood
estimate method, with a Bootstrap value set to 1000 and specific
parameters set to WAG+G. Construct the phylogenetic tree accordingly and
use the online beautification tool iTOL (Interactive Tree Of Life) to
enhance the visualization of the evolutionary tree.Then, according to
the selected genes, TBtools software was used to draw the heat map of
their expression in each tissue of longan.

## 3.3 Test

After performing a preliminary BLAST comparison between the amino acid
sequences predicted from related genes and the third-generation genome
sequence of longan, nine gene members were suspected to have homologous
relationships. However, after further analysis using DNAMAN software,
the results indicated that the identity between the exogenous gene and
the endogenous gene in longan was only 35.85%.

![Figure 3 Phylogenetic tree](https://static.igem.wiki/teams/5385/engineering/3.png)

Figure 3 Phylogenetic tree

![Figure 4  Expression of each gene in different tissues of longan](https://static.igem.wiki/teams/5385/engineering/4.png)

Figure 4 Expression of each gene in different tissues of longan

![Figure 5 Results of multiple sequence alignment](https://static.igem.wiki/teams/5385/engineering/5.png)

Figure 5 Results of multiple sequence alignment

## 3.4 Learning

Therefore, we preliminarily conclude that there are no relevant
homologous genes in longan, clarifying at the genetic level the reason
for the lack of astaxanthin content in longan. This also suggests that
the feasibility of increasing astaxanthin content by overexpressing
genes related to astaxanthin synthesis in longan is low.

# 4 Iteration Four Gene Transformation

## 4.1 Design

Based on the results of Iteration Three, we designed a plan to transfer
the exogenous genes, CrBKT from
Chlamydomonas reinhardtii and
HpBHY from
Haematococcus pluvialis, into
callus tissue. The objective is to explore the impact of these exogenous
genes on astaxanthin synthesis in longan, thereby validating the
feasibility of subsequent high-astaxanthin expression vectors in longan.

## 4.2 Build

We used pCAMBIA1301+CaMV35S+NOS vector modified by
pCAMBIA1301 vector to facilitate the regulation of initiation and
termination of transcription.By designing reverse primers, we performed
inverse PCR to linearize the vector. Using SnapGene software, we
designed primers for the target genes and employed seamless cloning
technology to ligate the
CrBKT gene extracted from
Chlamydomonas reinhardtii and
the HpBHY gene from
Haematococcus pluvialis into the
linearized pCAMBIA1301+CaMV35S+NOS vector. Next, the
recombinant gene was transformed into DH5α competent
E.coli cells, resuscitated, and
then coated in LB solid medium containing kanamycin for screening to
obtain recombiners.Finally, we verified the successful construction of
the recombinants through bacterial liquid PCR and sequencing.

![Figure 6 Reverse Primers](https://static.igem.wiki/teams/5385/engineering/18709c12-2d29-b5ee-9511-750f05a54bcb.jpg)

Figure 6 Reverse Primers

## 4.3 Test

### (1)Screening

**Screening Strategy:** Kanamycin is used to cultivate the
Escherichia coli after transformation, and a screening method is
employed to select recombinant colonies.

Confirmation Method: The large fragment recovered after performing
inverse PCR on the vector pCAMBIA1301-CrBKT-HpBHY is ligated with the
recovered gene fragment, and the ligation product is transformed into
E.coli. By selecting a culture
medium, the clones are tested for positivity. Further colony PCR reveals
that a single band of 3432bp is produced. This indicates that the
CrBKT gene and the
HpBHY gene have been
successfully cloned into the vector.

![Figure 6 Schematic diagram of colony PCR primers](https://static.igem.wiki/teams/5385/engineering/6.png)

Figure 7 Schematic diagram of colony PCR primers

### (2)Transformation, Antibiotic Preparation, and Agrobacterium Activation

Prepare 50 mg/ml of kanamycin sulfate and 25 mg/mL of rifampicin. Add
these antibiotics to LB liquid medium in a volume ratio of 1:1000,
respectively, and mix well.

Take the Agrobacterium competent cells and add 0.01-1 μg of plasmid DNA
per 100 μL of competent cells, mixing thoroughly. Allow the mixture to
stand on ice for 5 minutes, then sequentially place it in liquid
nitrogen for 5 minutes, followed by a 5-minute water bath at 37°C, and
finally another 5-minute ice bath. Subsequently, under a 28°C
environment, add 700 μL of LB liquid medium without antibiotics and
shake the culture for 150 minutes. Centrifuge to collect the bacteria
and retain some supernatant to resuspend the bacteria. Spread the
resuspended bacteria onto an LB plate containing kanamycin. Invert the
plate and incubate it in a 28°C incubator for 48 hours. The monoclonal
colonies were selected and transferred to LB liquid medium containing
kanamycin for extended culture at 28°C and 200 rpm for 24 hours.

### (3)Infection Process

Infect the well-grown embryogenic callus (EC) of longan with
Agrobacterium suspension at an
OD600 value of 0.6-0.8 for 30 minutes. Inoculate the infected callus in
small clusters and spread them evenly onto the culture medium for
cocultivation. After cocultivation on MS medium for 3 days, perform GUS
staining for identification. Subsequently, extract RNA separately to
identify positive transgenic cell lines, which will be utilized for
subsequent experimental research.

### (4)Inoculation

Remove the mixture of
Agrobacterium suspension and
longan callus from the shaker. After disinfecting or sterilizing all the
experimental equipment, proceed to work in Biosafety Cabinet.Pour the
bacterial solution and longan callus mixture from the centrifuge tube
into the filter paper funnel and let it sit for a few moments to allow
the bacterial solution to fully filter out.Inoculate the longan callus,
which has been drained of excess bacterial suspension, onto MS solid
medium. After sealing the plates, incubate them at 25°C in the dark for
3 days to promote the growth of the callus.

## 4.4 Learning

Based on this, we have introduced exogenous genes into the embryogenic
callus of longan through a series of procedures including gene cloning,
Agrobacterium-mediated
transformation, infection of the embryogenic callus, and inoculation and
cultivation. Preliminary and cultivation were also conducted, successfully
verifying the technical feasibility of establishing a high-expression
system for astaxanthin in longan.

# References

[1] Artimo.,Panu,Jonnalagedda.,Manohar,Arnold.,Konstantin,Baratin.,Delphine,Csardi.,Gabor,de,Castro.,Edouard,Duvaud.,Severine,Flegel.,Volker,Fortier.,Arnaud,Gasteiger.,Elisabeth,Grosdidier.,Aurelien,Hernandez.,Celine,Ioannidis.,Vassilios,Kuznetsov.,Dmitry,Liechti.,Robin,Moretti.,Sebastien,Mostaguir.,Khaled,Redaschi.,Nicole,Rossier.,Gregoire,Xenarios.,Ioannis,Stockinger.,&Heinz.(2012).ExPASy:SIBbioinformatics resource portal.NUCLEICACIDSRESEARCH,40(W1),W597-W603.

[2] Qianqian, D., & Yourui, X. (2023). Article title [Article title not
provided]. China Marine Medicine, 42(2), 69-76.
https://doi.org/10.13400/j.cnki.cjmd.2023.02.003

[3] Li, Y., Miao, F., Geng, Y., et al. (2012). Accurate quantification
of astaxanthin from Haematococcus crude extract
spectrophotometrically. Chinese Journal of Oceanology and Limnology,
30, 627-637. https://doi.org/10.1007/s00343-012-1217-5

[4] Xiaodan, W., Baoling, L., Yu, G., et al. (2021). Astaxanthin
synthesis of key genes in tobacco instantaneous expression and effect.
Journal of Shanxi Agricultural University (Natural Science Edition),
9(4), 33-40. https://doi.org/10.13842/j.carol.issn1671-8151.202101007

[5] Wang, F., Gao, B., Wu, M., Huang, L., & Zhang, C. (2019). A novel
strategy for the hyper-production of astaxanthin from the newly
isolated microalga Haematococcus pluvialis JNU35. Journal of
Hydrobiology, 510632. https://doi.org/10.1016/j.algal.2019.101466

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1 Carotenoid extraction experiments

1.1 Experimental preparation

1.2 Experimental operation

1.3 Data analysis

1.4 Precautions

![](https://static.igem.wiki/teams/5385/footer/footerline.png)

2 Astaxanthin extraction experiment

2.1 Experimental preparation

2.2 Experimental operation

2.3 Data analysis

2.4 Precautions

![](https://static.igem.wiki/teams/5385/footer/footerline.png)

3 Astaxanthin extraction experiment

3.1 Overview

3.2 Construction of target gene expression vector

3.3 Preparation of antibiotics

3.4 Transformation method

3.5 Activation of Agrobacterium tumefaciens

![](https://static.igem.wiki/teams/5385/footer/footerline.png)

References

# 1 Carotenoid extraction experiments

## 1.1 Experimental preparation

### (1) Experimental design

Carotenoids are fat-soluble pigments that are insoluble in water, but soluble in organic solvents such
as acetone, petroleum ether, and chloroform. Commonly used extraction methods include organic solvent
method, supercritical fluid extraction method, enzyme-catalyzed reaction method, and organic
solvent-based ultrasonic or microwave-assisted extraction method. In this experiment, carotenoid content
kit will be used to extract and quantitatively detect and carotenoids in plant tissues [1].

The principle of detection of carotenoids is based on their insoluble properties in water and soluble in
organic solvents. After crude extraction by organic solvents, the absorbance (A) of the solution can be
calculated using the Lambert-Beer law, which is proportional to the solute concentration (C) and the
thickness of the liquid layer (L), expressed as A = αCL, where α is the absorbance coefficient. In this
kit, carotenoids show a maximum absorption wavelength at 440. Based on the empirical formula, the
content of total and carotenoids can be calculated. This kit is mainly used in the field of scientific
research, especially for the extraction and quantitative detection of and carotenoids in plant tissues,
and is not suitable for clinical diagnosis or other purposes.

### (2) Preparation of experimental materials

Kit: The carotenoid content kit was purchased from Horui Corporation

Instruments: balance, oven, 100 mesh sieve, beaker, funnel, gauze, glass test tube, visible
spectrophotometer,microplate reader, trace quartz cuvettes, 96-well plate.

### (3) Reagent composition and preparation

1．Reagent 1: liquid (product solution formula not announced), 1000 mL × 2 bottles, store at 4°C.

2．Reagent 2: liquid (product solution formula not published), 1000 mL × 1 bottle, store at 4°C.

3．Extracting Solution: Mix reagent 1 and reagent 2 in proportion (2:1) before use, and seal and store at
4 °C to prevent volatilization.

## 1.2 Experimental operation

### (1) Sample handling

1. The longan fruit, peel, and pit (powder) are dried and crushed at 60 °C, and sieved through a 100
mesh sieve.

2. Weigh about 0.2 g of the sample and place it in a glass test tube.

3. Add 6 mL of the extract solution to the tube and extract for 20 minutes.

4. Filter the extract into a 50 mL centrifuge tubesand repeat the extraction until the residue is
colorless.

5. Combine the filtrate and set the volume to 50 mL to be measured.

![](https://static.igem.wiki/teams/5385/experiment/figure-1-longan-leaf.png)

Figure 1 Longan leaf

![](https://static.igem.wiki/teams/5385/experiment/figure-2-carotenoids-extracted-from-longan-leaves.png)

Figure 2 Carotenoids extracted from longan leaves

### (2) Absorbance measurement

1. Add the extract solution to the trace quartz cuvettes.

2. After zeroing with the extraction solution, the absorbance was determined by a microplate reader.

3. Absorbance recorded at 440 nm (A) respectively.

4. Calculate the absorbance difference:

$$ \Delta A = A\_{\text{measurement}} - A\_{\text{blank}} $$

[NOTE]:Blank tube only needs to be measured once

## 1.3 Data analysis

The formula for determination with a trace quartz cuvette is as follows:

$$ \text{Carotenoid content } (\mu g/g) = \frac{\Delta A \cdot V }{(\varepsilon \cdot d) \cdot W} \cdot
1000 = \frac{20 \cdot \Delta A}{W}
$$

V: total volume of extract, 50mL;

ε: Empirical absorbance coefficient of carotenoids, 2500;

d: cuvette aperture, 1cm;

W: Sample mass, g

## 1.4 Precautions

1. The extract solution is volatile and toxic, so take protective measures during operation.

2. The measurement must be rapid to prevent errors caused by volatilization.

# Astaxanthin extraction experiment

## 2.1 Experimental preparation

### (1) Experimental design

Astaxanthin is a kind of carotenoid, which is a fat-soluble pigment, insoluble in water, but soluble in
organic solvents such as acetone, petroleum ether, and chloroform. In this experiment, organic solvent
extraction method was used to extract and quantitatively detect astaxanthin-containing materials such as
longan leaves.

The detection principle of astaxanthin is based on its insoluble properties in water and soluble in
organic solvents. After crude extraction by DMSO, the absorbance of the solution ($A\_{530}$) can be
calculated using a calculation formula[2][3][4].

### (2) Preparation of experimental materials

Instruments: balance, oven, 100 mesh sieve, glass tube, microplate reader, 96-well plate,50 mL
centrifuge tubesand.

### (3) Reagent composition and preparation

DMSO, 500 mL × 1 bottle, and keep in constant temperature sealed away from light.

## 2.2 Experimental operation

### (1) Sample handling

1. Dry and crush the longan young leaves, longan old leaves and longan flesh at 60°C and sieve through a
100-mesh sieve.

2. Weigh about10 mg of the sample and place it in a glass tube.

3. Add 5mL of DMSO to the tube and extract for 10 minutes.

4. Centrifuge the mixture at 4,000 rpm for 5min and collect the supernatant.

5. Filter the extract into a 50 mL centrifuge tubesand, and repeat the extraction until the residue is
colorless.

6. Combine the filtrate and set the volume to 25 mL to be measured.

### (2) Absorbance measurement

1. Add the extract solution to 96-well plate.

2. After zeroing with DMSO, use the microplate method.

3. Absorbance ($ A\_{530} $) recorded at 530 nm respectively.

## 2.3 Data analysis

The formula is as follows:

$$ C\_A = \frac{A\_{530} - 0.0107}{0.1556} $$

$$ \text{Astaxanthin content } \left(\% \text{ of DW}\right) = \frac{0.025 C\_A \times 100\%}{m}
$$

Where $C\_{A}$ is the astaxanthin concentration (mg/L),

$A\_{530}$ is the absor bance value at 530nm,

$m (g)$ is the weight of the Longan tissue powder[4].

## 2.4 Precautions

1. The extract solution is volatile and toxic, so take protective measures during operation.

2. The measurement must be rapid to prevent errors caused by volatilization.

3. Away from light during extraction.

# 3 Vector construction

## 3.1 Overview

*HpBHY* and *CrBKT* were used as target genes to construct the
pCAMBIA1301-CrBKT-HpBHY
expression
vector. We
designed *CrBKT-HpBHY* seamless cloning primers according to the published sequences through
SnapGene
software, amplified the target gene by PCR, designed pCAMBIA1301+CaMV35S
+NOS reverse primers according
to the published vector sequences. The vector was linearized by reverse PCR and then obtained
pCAMBIA1301-CrBKT-HpBHY
after linking, transformation and screening.

## 3.2 Construction of target gene expression vector

The pCAMBIA1301-CrBKT-HpBHY expression vector was constructed using *HpBHY* from
*Haematococcus pluvialis*
and *CrBKT* from *Chlamydomonas reinhardtii* as the target genes.

1. Design *HpBHY-CrBKT* seamless cloning primers according to published sequences;

2. PCR amplification of the target gene according to the designed primers, and gel extraction of the
target gene band.

3. The reverse primer of pCAMBIA1301+CaMV35S+NOS was designed according to the published vector
sequence, and the 1301 was linearized at no load and gel extraction of the linearized carrier band.

4. Ligation via T4 DNA ligase overnight at 16°C; DH5α competent was transformed and plated into
Kana-containing LB solid medium, monoclonal was selected and colony PCR was performed, and finally the
pCAMBIA1301-CrBKT-HpBHY
recombinant vector was obtained.

![](https://static.igem.wiki/teams/5385/engineering/18709c12-2d29-b5ee-9511-750f05a54bcb.jpg)

Figure 3 Reverse Primers

![](https://static.igem.wiki/teams/5385/experiment/figure-3-electrophoretic-sampling-was-performed.png)

Figure 4 Electrophoretic sampling was performed

## 3.3 Preparation of antibiotics

50mg/mL kanamycin sulfate was prepared by mixing kanamycin sulfate 0.5 g with 100mL double distilled
water. Rif 0.25 g and 100 mL Dimethyl sulfoxide were used to prepare 25 mg Rifampicin antibiotics.
50mg/mL kanamycin sulfate and 25mg/mL rifampicin antibiotics were added into LB liquid medium at the
volume ratio of 1:1000 and mixed.

## 3.4 Transformation method

1. *Agrobacterium* receptor state stored at -80℃ needs to be slightly melted at room temperature
or in the hand, and inserted into the ice when in the mixed state of ice and water.

2. Add 0.01-1 μg of plasmid DNA per 100 μL of competent (To improve transformation efficiency, we
performed a pre-experiment to determine the plasmid amount before first use), gently dial the bottom of
the tube and mix well, and let it stand on ice for 5 minutes, liquid nitrogen for 5 minutes, a 37°C
water bath for 5 minutes, and finally an ice bath for 5 minutes.

![](https://static.igem.wiki/teams/5385/experiment/figure-4-thermostatic-water-bath.png)

Figure 5 Thermostatic water bath

3. 700 μL of antibiotic-free LB liquid medium was added to the mixture and cultured at 28℃ for 2-3
hours.

4. *Agrobacterium* were collected at 6000 rpm centrifugation for 1 min, retaining about 100 μL of
supernatant and lightly beating the resuspended block, and then coated on LB plates containing the
corresponding antibiotics, culture was inverted in a 28 ℃ incubator for 2-3 days;

5. The monoclonal colonies were selected and transferred to liquid medium for extended culture.

6. *Agrobacterium* solution was added into the LB medium containing antibiotics, the sealing film
is sealed, and the mixed bacterium liquid is oscillated. The bacteria was shaked in shaking bed
vibration culture at 180 rpm under 28℃ for 2d.

*Agrobacterium*, as an aerobic bacterium, needs a lot of oxygen to grow. Successful liquid shakes
require sufficient dissolved oxygen in the medium:

(1) Use triangular bottles;

(2) Keep the cross-section of the culture medium large and the thickness small;

(3) Add antibiotics as little as possible, and use low concentrations when they must be added.

## 3.5 Activation of Agrobacterium tumefaciens

### 3.5.1 Adjust the OD value of Agrobacterium

**(1) Preservation of bacterial solution**

500 μL of activated *Agrobacterium* was transferred into the cryopreservation tube, 60% glycerol
was added
into the same volume, and then the liquid nitrogen was quickly frozen and stored in the ultra-low
temperature refrigerator at -80 ℃.

**(2) Centrifuge**

The remaining bacterial solution was balanced and placed in a centrifuge at 28 ℃ and 5000 rpm for 10
minutes.

![](https://static.igem.wiki/teams/5385/experiment/figure-5-centrifugal.png)

Figure 6 Centrifugal

![](https://static.igem.wiki/teams/5385/experiment/figure-6-centrifugal.png)

Figure 7 Centrifugal

**(3) Preparation of culture medium**

At the volume ratio of 1:10000, 50 mmol/L acetosyringone and 10 mmol/L MgCl2 were added into MS liquid
medium in turn, and shaken to mix.

**(4) Suspension precipitation**

After centrifugation, the supernatant was discarded. The MS Medium and the mixed solution of
acetosyringone and MgCl2 were added into the sediment, beat well, re-suspension of the block.

**(5) Cuvette treatment**

Prepare 5 clean trace quartz cuvettes, rinse twice with distilled water and once with re-suspension to
ensure no impurities in trace quartz cuvettes.

**(6) Determination of absorbance**

In the super-clean working table, the suspension was added to the trace quartz cuvettes, another one was
taken out and rinsed, and MS liquid medium was added as the control. UV photometer was used to determine
absorbance at 600 nm and the OD value was adjusted to 0.6-0.8.

**(7) Culture treatment**

The modified OD value of *Agrobacterium* was cultured in a dark shaker at 28℃ and 180 rpm for 1
hour in
order to prepare for subsequent experiments.

### 3.5.2 Infection

*Agrobacterium* tumefaciens with OD600 of 0.6-0.8 was used to infect EC of embryogenic calli of
longan with good growth condition for 30 min. After infection, longan EC was filtered and sucked up the
residual bacterial liquid, and inoculated in the medium in a small round way for co-culture. After
co-culture on MS Medium for 3 days, GUS staining was performed, identification, RNA was extracted
separately for identification of positive transgenic cell lines, and positive transgenic cell lines were
added to the liquid medium shaker for later use.

![](https://static.igem.wiki/teams/5385/experiment/figure-7-callus.png)

Figure 8 Callus

### 3.5.3 Inoculate

**(1) Preparation**

Fold the filter paper into a funnel shape and place it on the flask. Flasks and parafilm are sterilized
with 75% alcohol and then placed in a sealing film for UV sterilization to ensure a sterile environment.

![](https://static.igem.wiki/teams/5385/experiment/figure-8-sterilization-operation.png)

Figure 9 Sterilization operation

**(2) Separation**

Remove the *Agrobacterium* liquid centrifuge tube containing the longan callus from the shaker.
Pour the bacterial solution and longan callus mixture from the centrifuge tube into the filter paper
funnel and let it sit for a few moments to allow the bacterial solution to fully filter out.

![](https://static.igem.wiki/teams/5385/experiment/figure-9-shake-the-bacteria.png)

Figure 10 Shake the bacteria

![](https://static.igem.wiki/teams/5385/experiment/figure-10-shake-the-bacteria.png)

Figure 11 Shake the bacteria

**(3) Drain**

When there is not much bacteria solution left in the funnel, use a medicine spoon to carefully remove
the longan callus, place it on a clean filter paper sterilized by the flame of an alcohol lamp, and
drain it to make the bacteria on the material fully absorbed by the filter paper.

**(4) Inoculation**

The longan calli of the filtten solution were inoculated on MS solid medium, sealed and placed in a dark
culture at 25°C for 3 days to promote the growth of callus.

# References

[1] Qianqian, D., & Yourui, X. (2023). Article title [Article title not provided]. China Marine
Medicine, 42(2), 69-76. https://doi.org/10.13400/j.cnki.cjmd.2023.02.003

[2] Li, Y., Miao, F., Geng, Y., et al. (2012). Accurate quantification of astaxanthin from Haematococcus
crude extract spectrophotometrically. Chinese Journal of Oceanology and Limnology, 30, 627–637.
https://doi.org/10.1007/s00343-012-1217-5

[3] Xiaodan, W., Baoling, L., Yu, G., et al. (2021). Astaxanthin synthesis of key genes in tobacco
instantaneous expression and effect. Journal of Shanxi Agricultural University (Natural Science
Edition), 9(4), 33-40. https://doi.org/10.13842/j.carol.issn1671-8151.202101007

[4] Wang, F., Gao, B., Wu, M., Huang, L., & Zhang, C. (2019). A novel strategy for the hyper-production
of astaxanthin from the newly isolated microalga Haematococcus pluvialis JNU35. Journal of Hydrobiology,
510632. https://doi.org/10.1016/j.algal.2019.101466

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Overview

1 Detection of Carotenoid Content in Various Tissues of Wild Longan

1.1 Result Analysis

![](https://static.igem.wiki/teams/5385/footer/footerline.png)

2 Detection of Astaxanthin Content in Wild-Type Dimocarpus longan
Tissues

3 Successful Construction of the Vector pCAMBIA1301-CrBKT-HpBHY

3.1 Result Analysis

3.2 Infection

![](https://static.igem.wiki/teams/5385/footer/footerline.png)

References

# Overview

In this section, we present and analyze the results obtained from the engineering part. Through
detection, we determined the carotenoid and astaxanthin contents in wild-type *Dimocarpus longan*, and
based on this, inferred the feasibility of constructing a high-astaxanthin-expressing system in longan.
Following this, based on the chemical synthesis pathway of astaxanthin, we successfully constructed the
pCAMBIA1301-CrBKT-HpBHY vector as the foundation.

*Agrobacterium*-mediated transformation technology was used to introduce it into longan callus, hoping to
culture the longan plants with high astaxanthin expression and explore its application potential in
Longan production.

# 1 Detection of Carotenoid Content in Various Tissues of Wild Longan

Since astaxanthin and its precursors belong to the carotenoid family, we use the carotenoid content in
longan as a direct indicator to assess the potential for astaxanthin production in various tissues of
longan. To this end, we sampled five tissues from wild-type longan, including old leaves, young leaves,
pericarp, seed, and pulp, with three biological replicates and technical replicates for each tissue.
Carotenoids were extracted and the results are as follows.

![](https://static.igem.wiki/teams/5385/results/figure-1-histogram-of-carotenoid-content.png)

Figure 1 Histogram of carotenoid content

## 1.1 Result Analysis

The highest carotenoid content was found in older leaves, consistent with their role in
photosynthesis[4]. Given that carotenoids such as astaxanthin are synthesized to protect plants from
oxidative stress, the high levels in mature leaves suggest that they are crucial for photoprotection and
chlorophyll stabilization[3]. This tissue may be a potential focus for further astaxanthin
production[1].

Although carotenoid content in young leaves is lower than in mature leaves, they still show a
significant amount of carotenoids, which may indicate active biosynthesis during early developmental
stages. The growth process from young to mature leaves provides valuable insights into the regulatory
pathways of carotenoid biosynthesis, which is important when considering astaxanthin production.

The moderate carotenoid content in the fruit peel indicates its role in protecting the fruit from
environmental factors such as UV radiation and pests. Carotenoids in the peel may contribute to the
plant's defense mechanisms[2]. Compared to leaf tissues, the peel may be less ideal for astaxanthin
production but could still serve as a secondary source[3].

The carotenoid content in the seeds is negligible, making them unlikely to be a viable raw material for
astaxanthin extraction.

The carotenoid content detected in the fruit pulp is minimal, indicating that extracting astaxanthin
directly from the fruit may not be highly satisfactory. However, given the edible value of the fruit,
increasing its astaxanthin content still holds potential and significant scientific importance.

This preliminary data suggests that focusing on leaves may produce the greatest amounts of astaxanthin.
Further research could explore optimizing astaxanthin extraction methods or enhancing the specific
biosynthetic pathways of astaxanthin in *Dimocarpus longan* tissues.

# 2 Detection of Astaxanthin Content in Wild-Type Dimocarpus longan Tissues

Unlike the potential for increasing astaxanthin content indicated by the tissues responding to
carotenoid detection, this analysis directly measures the percentage of astaxanthin relative to the dry
weight of wild-type *Dimocarpus longan*, providing a direct representation of the astaxanthin
content in
wild-type *Dimocarpus longan* to explore its scientific value.

![](https://static.igem.wiki/teams/5385/results/figure-2-histogram-of-astaxanthin-content.png)

Figure 2 Histogram of astaxanthin content

The extraction results indicate that the astaxanthin content in wild-type *Dimocarpus longan* is
extremely
low. Due to this minimal amount, the experimental results are significantly affected by the precision of
the experimental equipment, resulting in a low reliability of the specific numerical values. However, it
can still be inferred that wild-type *Dimocarpus longan* plants may contain either very low
concentrations
of astaxanthin or none at all, suggesting that there is no value in directly extracting astaxanthin from
wild-type *Dimocarpus longan*.

Nevertheless, the carotenoid extraction experiments from wild-type *Dimocarpus longan* indicate
that the
carotenoid content is considerable. Based on *Dimocarpus longan*, there is theoretical
feasibility and
technical direction for constructing a high-expression astaxanthin system through the extraction of
carotenoids and utilizing synthetic biology.

# 3 Successful Construction of the Vector pCAMBIA1301-CrBKT-HpBHY

We used SnapGene software to design primers according to the sequence of pCAMBIA1301-CrBKT-HpBHY, and
confirmed
whether the vector pCAMBIA1301-CrBKT-HpBHY was successfully constructed by colony PCR.

PCR reaction system

| Constituent | Volume |
| 2x Taq polymerase | 5.0 l |
| Forward primer | 0.5 l |
| Reverse primer | 0.5 l |
| Bacterial solution | 1.0 l |
| & ddH\_{2}O & | 3.0 l |
| Total volume | 10.0 l |

We transformed the plasmid into DH5α-receptive *E.coli*. PCR identification was performed to confirm the
construction of vector pCAMBIA1301-CrBKT-HpBHY. The results are as follows:

![](https://static.igem.wiki/teams/5385/results/figure-3-electrophoresis-results.png)

Figure 3 Electrophoresis results

## 3.1 Result Analysis

Colony PCR was performed on the obtained *E.coli* according to the primers designed by us. After PCR
amplification and electrophoresis, if the vector was successfully constructed, the corresponding
fragment CrBKT-HpBHY was amplified by PCR, and the size of the PCR product should be 3432 bp. If the
construction is not successful, there is no PCR band.

![](https://static.igem.wiki/teams/5385/results/figure-4-pcambia1301-camv35s-nos-empty-vector.png)

Figure 4 pCAMBIA1301+CaMV35S+NOS empty vector

![](https://static.igem.wiki/teams/5385/results/figure-5-pcambia1301-crbkt-hpbhy-vector.png)

Figure 5 pCAMBIA1301-CrBKT-HpBHY vector

![](https://static.igem.wiki/teams/5385/results/figure-6-colony-pcr-primers.png)

Figure 6 Colony PCR Primers

The electrophoresis results showed that the band is located between 3000 bp and 4000 bp. This confirms
that we have successfully constructed the vector pCAMBIA1301-CrBKT-HpBHY and successfully transformed
the gene pCAMBIA1301-CrBKT-HpBHY into the DH5α-competent *E.coli*

## 3.2 Infection

At present, we are using *Agrobacterium* to infect longan callus.

![](https://static.igem.wiki/teams/5385/results/figure-7-callus.png)

Figure 7 Callus

Our future plans are as follows:

**1.Observation of Callus Growth**

We will regularly monitor the growth of the *Dimocarpus longan* embryogenic callus tissue on the MS solid
medium to ensure that it remains in good condition. If abnormal growth or contamination is observed,
appropriate measures, such as changing the medium or adjusting growth conditions, will be taken
promptly.

**2.Molecular Verification**

Once the callus growth is stable, we will perform PCR analysis for verification at the gene level.

**3.Gene Expression Verification in Transgenic Plants**

Finally, we will conduct further molecular analysis, such as Western blot, to confirm the expression
level of the target gene. If successful, we will evaluate the gene's functional performance to
understand its specific effects on *Dimocarpus longan*.

# References

[1] Demmig-Adams,B,Gilmore,A,M,Adams,W,& W.(1996).Carotenoids 3: in vivo function of carotenoids in
higher plants..FASEB J,10(4),403-12.

[2] Rojas-Garbanzo C, Gleichenhagen M, Heller A, Esquivel P, Schulze-Kaysers N, Schieber A. Carotenoid
Profile, Antioxidant Capacity, and Chromoplasts of Pink Guava (Psidium guajava L. Cv. 'Criolla') during
Fruit Ripening. J Agric Food Chem. 2017 May 10;65(18):3737-3747. doi: 10.1021/acs.jafc.6b04560. Epub
2017 May 2. PMID: 28301727.

[3] Li H S. (2018). Effects of light on functional metabolites in longan cell culture and its molecular
mechanism.

[4] Zhou, D., Fang, Z., Liao, R., Ye, X., Jiang, C., & Pan, S. (2013). Study on the contents and
antioxidant properties of anthocyanins, flavonoids, and carotenoids in plum peel. Chinese Journal of
Nutrition, 35(6), 571-576.

[5] Leydesdorff, L., & Rafols, I. (2009). A global map of science based on the ISI subject categories.
Journal of the American Society for Information Science and Technology, 60(2), 348-362.

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