iGEM 2022 — Team fafu-china

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2022.4.18

DlBADH2、DlGAD-2、DlGAD3-2、DlGAD-5 construct 1301 vector

2022.4.27

DlCaM-5、DlCaM-8、DlCaM-25、DlCaM-31、DlGAD construct 1301 vector

2022.5.16

DlGAD-2、DlGAD-5 construct 1301 vector

2022.7.3

Determination of GABA in longan callus(7d、10d、12d)

2022.7.5

Construction of CaM vector for gene editing pGW3:030、025

2022.7.9

DlGAD-2、DlGAD-5 construct 1301 vector

2022.7.10

Substitute 50 bottles of medium without adding silver

2022.7.15

Subculture 29 bottles of materials

2022.7.19

DlGAD-2、DlGAD-5 construct 1301 vector

2022.7.20

Transferring CaM vector of pGW3 into Agrobacterium tumefaciens

2022.7.25

Agrobacterium tumefaciens liquid PCR

2022.7.29-7.31

SubcultureR

2022.8.13

Agrobacterium infection of longan callus

2022.8.14-8.15

SubcultureR

2022.8.20

Transgenic material transfer and sample collection

2022.9.1

SubcultureR

2022.9.6

SubcultureR

2022.9.7

Transgenic material reverse transcription

2022.9.14

Real time quantitative PCR

2022.9.24

SubcultureR

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![engineering](https://static.igem.wiki/teams/4113/wiki/wiki-image/background-image/engineering.png)

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# Overview

    We conducted experiments in the lab strictly according to the
"design-build-test-learn" cycle. To achieve the goal of building a high-yield GABA longan cell
factory, we first conducted the design of experiments, mainly by reviewing a large amount of
literature to determine the appropriate GABA synthesis pathway and using HMM search. In the
process, we created five composite parts, one of which was created based on the existing basic
parts, and two parts were not available due to the confidentiality principle. We then
transferred the vector to Agrobacterium tumefaciens-infested longan healing tissues and treated
them with both blue light and high temperature to test whether the system we constructed could
achieve a real increase in GABA content in longan healing tissues and form a long-term stable
succession culture of high-yielding GABA cell system to expand the culture using fermenters.
After passing the test, we will reflect and summarize our experience from the above three parts
to better carry out the next ground engineering cycle and expect to extract the GABA from the
high GABA-yielding longan cell line to better serve the pharmaceutical industry and achieve our
ultimate goal.
![engineering26](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering1.png)
Fig.1 iGEM engineering cycle
![engineering27](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering2.png)
Fig.2 Engineering Cycle Diagram

# Identification of genes

Determination of the pathway

    In higher plants, the metabolism of GABA is mainly accomplished by three
enzymes, firstly, the
irreversible decarboxylation of L-glutamic acid (Glu) at the α-position under the action of GAD
to form GABA, and then the reaction of GABA with pyruvate and α-ketoglutarate catalyzed by GABA
transaminase (GABA-T) to form succinate. Then, GABA reacts with pyruvate and α-ketoglutarate to
form succinic semialdehyde, and finally succinic semialdehyde dehydrogenase (SSADH) catalyzes
the oxidative dehydrogenation of succinic semialdehyde to form succinic acid, which finally
enters the tricarboxylic acid cycle (krebs circle). This metabolic pathway constitutes a branch
of the TCA cycle, called the GABA branch.
![engineering1](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering3.png)
Fig3. Plant GABA synthesis pathway
    After comparing the three synthesis pathways, we chose the pathway of
GABA generation from glutamate catalyzed by GAD in the GABA branch.

GAD&CaM gene screening

    By means of sequence alignment, phylogenetic affinity analysis,
structural domain validation and transcriptome analysis, we successfully screened
DlGAD2,DlGAD5,DlCaM1 and DlCaM2 as candidate genes for constructing the vector system.
![engineering3](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering4.png)
Fig.4 Evolutionary tree of CaM genes in longan, Oryza sativa L. and
Arabidopsis
![engineering4](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering5.jpg)
Fig.5 Conservative structural domain of CaM genes in longan, Oryza sativa
L. and Arabidopsis.
![engineering5](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering6.jpg)
Fig.6 Expression of DlCaM in the early stage of somatic embryogenesis in
Longan
GAD gene screening
![engineering5](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering7.jpg)
Fig.7 Conservative structural domain of DlGAD genes in longan
![engineering5](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering8.jpg)
Fig.8 Expression of DlGADs in the early stage of somatic embryogenesis in
Longan
    During the previous screening of genes, we took many detours, such as
initially misclassifying DlCMLs within DlCaMs and not screening DlGADs precisely enough.
However, after correcting the mistakes one by one, we finally identified the target genes.

# Construction of vector

    We selected the pCAMBIA1301SN vector after modification of pCAMBIA1301
to construct the Longan
DlGAD overexpression vector, and we selected the pGW3 (Genovo-w-3,
Source: The New
Zealand Institute for Plant and Food Research Limited) vector to construct the sgRNA
of Longan DlCaM gene editing vector.

Construction of pCAMBIA1301SN-DlGAD overexpression
vector

    The constructed recombinant plasmid pCAMBIA1301SN was used as a template
for PCR using specific
primers for the DlGAD2 gene and DlGAD5 gene of Longan, and the results were shown in Fig10 and
Fig11. The sizes of PCR amplification products were about 1428bp and 1473bp, which were
consistent with the sizes of DlGAD2 gene and DlGAD5 gene of Longan.

![engineering8](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering9.png)
Fig.9 Flow chart of constructing Longan gene overexpression
vector(1)
![engineering9](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering10.png)
Fig.10 Flow chart of constructing Longan gene overexpression
vector(2)
    In order to further verify the accuracy of the insertion site of the
DlGAD2 gene in Longan, the
recombinant plasmid pCAMBIA1301-DlGAD2 was verified by double digestion using the restriction
endonucleases FastDigest KpnⅠ and FastDigest SalⅠ, and the recombinant plasmid
pCAMBIA1301SN-DlGAD5 was verified by double digestion using the restriction endonucleases
FastDigest KpnⅠ and FastDigest PstⅠ, and the digested products were imaged by electrophoresis
gel, and four bands were present as shown in Fig12. The serial number ① represents the band of
the product after double digestion of DlGAD5 with a length of 1473bp, serial number ② represents
the band of the product after double digestion of DlGAD2 with a length of 1428bp, serial number
③represents the band of the product after double digestion of the empty plasmid pCAMBIA1301SN
with a length of 12596bp by the restriction endonucleases FastDigest KpnⅠ and The band of the
product of double digestion of FastDigest PstⅠ and FastDigest SalⅠ by restriction endonucleases
FastDigest KpnⅠ and FastDigest PstⅠ, and the band of the product of double digestion of
pCAMBIA1301SN by restriction endonucleases FastDigest KpnⅠ and FastDigest SalⅠ, indicating the
correct insertion site of DlGAD2 and DlGAD5 on the vector. The insertion site was correct. The
results of the combined PCR assay and double digestion validation showed that the DlGAD5 and
DlGAD2 genes were successfully inserted into the polyclonal site of the intermediate expression
vector pCAMBIA1301SN and could be used for subsequent studies.
![engineering10](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering11.jpg)
Fig.11:PCR amplification product of DlGAD2
![engineering11](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering12.jpg)
Fig.12 PCR amplification product of DlGAD5
![engineering12](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering13.jpg)
Fig.13 Enzyme-digested product
Construction of pGW3-ΔDlCaM

    The basic flow of this section is as follows:
![engineering13](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering14.png)
Fig.14 Flow chart of constructing pGW3-ΔDlCaM
①Plasmids

The vector we used in our experiments was Genovo-W-3, commonly referred to as pGW3. pGW3 has a
fragment length of 16,654 bp. We cleaved it with BsaⅠ and gum recovered a fragment of size
15,429 bp, the pGW3 backbone. We used T4 ligase to ligate the sgRNA to the pGW3 backbone to
obtain the recombinant plasmid.
![engineering14](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering15.png)
Fig.15: Genovo-W-3 plasmid mapping
②Enzyme digestion

Plasmid pGW3 was digested with Bsa I:
![engineering15](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering16.jpg)
Fig.16 Enzyme-digested product
③Identification of positive clones

After 10 -12h, single clones were selected for colony PCR identification and sequencing to
confirm whether the target was attached to the vector. The bacteriophage with correct PCR bands
will be selected and a tube of large shake extracted plasmids will be sequenced.
![engineering16](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering17.jpg)
Fig.17 Bacteriophage PCR result
④Transfer of recombinant plasmid into Agrobacterium

![engineering17](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering18.jpg)
Fig.18: pGW3-ΔDlCaM1 recombinant vector and pGW3-ΔDlCaM2 recombinant
vector Agrobacterium
bacteriophage PCR

# Agrobacterium infestion

    Due to the epidemic, our access to the laboratory was somewhat hampered
during the summer and the culture of the material took longer, with the first successful
infestation only in mid-August and one in September. The infestation process requires a lot of
attention, and one mistake may cause material contamination leading to experimental failure, we
experienced many failures, and finally achieved success in the case of transient 6 days.

# Exogenous treatments

1.Blue light treatment

Material: Longan callus (+ Ag) subcultured for 20 days was
directly treated with blue light in
culture flask

Number of treatments: 27 bottles of materials with
relatively consistent growth

Blue light parameters: 32umol·m-2·S-2

Treatment time: 30min, 60min, 90min, 120min, 150min,
180min, 210min. Take out 3 bottles at each
time point mix the samples on the filter paper, put them into the freezing tube, make
corresponding marks, put them into liquid nitrogen, and store them in the refrigerator at -80 ℃.

The specific method was as follows: the materials treated
for 20 days were transferred with
black bags and placed in a blue light incubator, and the samples were mixed every 3 bottles when
they were collected. The materials treated by blue light at different times were collected 0.1g
per tube, and 6 tubes were collected respectively, and then put into liquid nitrogen immediately

2.High temperature stress treatment:

Material: Longan callus (+ Ag) subcultured for 20 days was
directly treated with high
temperature stress in culture flask

Number of treatments: 30 bottles of materials with
relatively consistent growth

Treatment temperature: 40 ℃

Treatment time: 30min, 60min, 90min, 120min,150min,
180min. Take out 3 bottles at each time
point, mix the samples on the filter paper, put them into the freezing tube, make corresponding
marks, put them into liquid nitrogen, and store them in the refrigerator at -80 ℃.

The specific method was as follows: the materials treated
for 20 days were transferred with
black bags and placed in the temperature treatment incubator, and the samples were mixed every 3
bottles when the samples were collected. The materials treated at high temperature for different
times were collected 0.1g per tube, and 6 tubes were collected respectively, and then put into
liquid nitrogen immediately

# GABA content

High temperature and blue light treatment

By observing the trend of GABA content change with time under different culture environments, we
found that the GABA content changed with time approximately normally distributed, and the GABA
content reached a higher level when the time reached about 30 min.
![engineering22](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering19.png)
Fig.21: Blue light processing
![engineering23](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering20.png)
Fig.22 High temperature treatment
GABA content in longan after knockdown of DlCaM1

![engineering24](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering21.png)
Fig.23 GABA content in longan after knockdown of DlCaM1
    As shown in the Fig., the endogenous GABA content of longan healing
tissues was increased
after knockdown of DlCaM1.

# Transfer to fermentor

    Under the influence of the COVID-19, we were not able to enter the
laboratory on time for the
experiments. In the last crunch, we decided to simulate the fermenter culture with suspension
culture of longan healing tissue, and the GABA content of the suspension culture material was
measured for 6 d of continuous sample collection, and the results are shown below:
![engineering25](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering22.png)
Fig.24 GABA content of suspension culture material
![engineering26](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering23.png)
Fig.25 Predicted content of GABA per yield
![engineering27](https://static.igem.wiki/teams/4113/wiki/wiki-image/engineering/engineering24.png)
Fig.26 Predicted annual GABA production content
    The GABA content of longan healing tissues cultured in suspension showed
an overall trend of
increasing and then slowly decreasing from day 6 to day 11 of the initial culture, and reached a
peak of 6440.567926 μg/g at day 7 of the suspension culture. The annual yield of longan healing
tissue in 5L and 30L fermenters was estimated. The single yield of 5L was 30447784.87 μg and the
annual yield was 1587634497 μg, and the single yield of 30L was 182686709.23 μg and the annual
yield was 9525806981.3 μg. Depending on this conclusion, the production of high yield GABA cell
factories can be guided as a way to increase the GABA content.

![longchild](https://static.igem.wiki/teams/4113/wiki/wiki-image/animation/a3.gif)

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click here

content

* GMO Engineering
* Gene Editing
* Determination of GABA Content
* Longan tissue culture
* Agrobacterium infestation

![smallLongGan](https://static.igem.wiki/teams/4113/wiki/wiki-image/animation/a4.gif)

Methods and Procedures

## Preparation of materials

1 Preparation of materials
Nutrient medium
Medium1:MS+1.0 mg/L 2, 4-D+20 g/L sucrose+7g/L Agar
Medium2:MS+1.0 mg/L2, 4-D+0.5 mg/L KT+0.5 mg/L Ag NO3+20 g/L
sucrose MS+2, 4-D+7g/L Agar
Material cultivation
Callus of Longan were subcultured alternately on Medium 1 and Medium 2.

## Construction of overexpression vector

2.1 RNA extraction from plant material samples
      1.After weighing the ultra-low temperature frozen RNA
extracted samples, they were quickly
transferred to a mortar and pestle pre-cooled with liquid nitrogen, and the tissues were
ground thoroughly with a mortar and pestle until they were ground into a powder, during
which time liquid nitrogen could be added. The yield and quality of the RNA can be affected
if it is not ground thoroughly.
      2.The samples ground to powder form were transferred to
a centrifuge tube and 1 ml of
TransZol Up was added for every 50-100 mg of tissue. homogenization was performed with a
homogenizer and repeated blowing with a gun.
      3.Allow to stand at room temperature for 5 minutes.
      4.For each 1 ml of TransZol Up used, add 200 ul of
chloroform, shake vigorously for 30
seconds and incubate at room temperature for 3-5 minutes.
      5.The sample was centrifuged at 10,000xg for 15 minutes
at 4°C. At this point the sample was
divided into three layers, a colorless aqueous phase (top layer), an intermediate layer, and
a pink organic phase (bottom layer). At this point the sample was divided into three layers,
a colorless aqueous phase (upper layer), an intermediate layer, and a pink organic phase
(lower layer.) The RNA was mainly in the aqueous phase, and the volume of the aqueous phase
was approximately 50% of the TransZol Up reagent used.
      6.Transfer the colorless aqueous phase to a new
centrifuge tube, add 500 ul of isopropanol
per 1 ml of TransZolUp used, mix upside down, and incubate in the greenhouse for 10
minutes.
      7.Centrifuge at 10,000 x g for 10 min at 4°C. Remove the
supernatant and form a gelatinous
precipitate on the side and bottom of the tube.
      8.Add l m1 75% ethanol (DEPC-treated water preparation)
and vortex vigorously (add at least
1 ml of 75% ethanol for every 1 ml of TransZol Up used).
      9.Centrifuge at 7500xg for 5 min at 4°C.
      10.Discard the supernatant (for better control of salt
ion content in RNA, ethanol should be
removed as much as possible), and dry the precipitate at room temperature (1-2 min).
      11.The precipitate is dissolved in 10-15 ul of RNA lysis
solution.
      12.The samples were stored at -70°C.

2.2 Reverse transcription cDNA system
![](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-experiment/experiment1.png)

2.3 Polymerase Chain Reaction(PCR)

![](https://static.igem.wiki/teams/4113/wiki/wiki-image/animation/sticky.png)

| Component | Volume |
| --- | --- |
| Green Enzyme Mix | 12.5μL |
| Upstream primers | 1μL |
| Downstream primers | 1μL |
| cDNA | 1μL |
| ddH2O | 9.5μL |

| Temperature (℃) | Time |
| --- | --- |
| 95 | 60s |
| 95 | 20s |
| 57 (adjustable) | 20s |
| 72 | 1min/kb |
| 72 | 5min |
| 4 | ∞ |

2.4 Electrophoresis
①Material
0.17g Agarose + 0.8μL Nucleic acid dye + 20ml 1×TAE
![](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-experiment/experiment2.jpg)
②Method
    Agarose, nucleic acid dye and 1×TAE were added to the conical flask,
heated for 2min and
poured into the plate, and a comb was inserted. After solidification, add sample,
electrophoresis 150V 100A, 20min

2.5 Gum recovery
All centrifugations were performed at room temperature
      1. Cut the target DNA strip from the agarose gel and put
it into a clean centrifuge tube to
weigh, if the gel weighs 100 mg, it can be regarded as 100 μL, and so on.
      2. Add 3 times the volume of solution GSB, melt the gel
in 5SC water bath for 6-10 minutes,
intermittently (2-3 minutes) mix to ensure the gel is completely melted, when the gel is
completely melted, observe the color of the solution, if the color is purple, add
appropriate amount of 3 M sodium acetate (pH5S2), adjust the color to be the same as GSB
color (yellow). (To increase the amount of DNA recovery, add 1x volume of isopropanol to the
melted gel solution, e.g. 100 mg of gel weight, add 100 μl of isopropanol).
      3. to be melted gel solution to room temperature (high
temperature centrifugation column
binding DNA ability is weak), added to the centrifugation column to stand for 1 minute,
1000xg centrifugation for 1 minute, discard the effluent.
      4. Add 650μl of solution WB, centrifuge at 10.000xg for
1 minute, discard the effluent.
      5. Centrifuge at 10,000\*g for 1-2 minutes to completely
remove the residual WB.
      6. Place the centrifugal column in a clean centrifuge
tube, open the cap and let it stand
for 1 minute to make the residual ethanol evaporate cleanly, add 30-50μl of EB or deionized
water (pH>7.0) in the center of the column (EB or deionized water preheated in a water bath
at 60-70C is better for use) and let it stand for 1 minute at room temperature.
      7. Centrifuge at 10000xg for 1 min, elute DNA, and store
the eluted DNA at -20°C.
![](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-experiment/experiment3.jpg)

Caution
      1.To ensure the recovery effect, use fresh
electrophoresis buffer when electrophoresis.
      2.When cutting the gel, make sure the gel is as small as
possible; when melting the gel,
make sure the gel is completely melted.
      3.To avoid damage to DNA caused by UV irradiation, which
may affect downstream experiments
(e.g. cloning, ligation), keep the UV irradiation time as short as possible.

2.6 Connection and Transformation
①LB Formulation
      LB broth 25g (preparation of liquid LB)
      LB nutrient agar 40g (prepared solid LB)
      Distilled water 1000ml
      Dissolve LB in distilled water, dispense, sterilize at
121℃ and set aside.
![](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-experiment/experiment4.jpg)
②Connection System

| Component | Volume |
| --- | --- |
| T4 DNA ligase Buffer | 2 μL |
| T4 DNA ligase | 1 μL |
| 1301/1302 Gel recovery product after emptying enzyme digestion | 4 μL |
| The target gene fragment after enzyme digestion | 13 μL |

③Transformation
      a. DH5α competent cells were taken out from -80℃,
quickly inserted into ice, and after 5
minutes, the bacterial block was thawed, the target DNA (plasmid or ligation product) was
added, and the bottom of the EP tube was gently mixed by hand to avoid using a gun Suck and
let stand in ice for 25 minutes.
      b. Heat shock in a 42℃ water bath for 45 seconds,
quickly put back on ice and let stand for
2 minutes, shaking will reduce the transformation efficiency.
      c. Add 700μL of antibiotic-free sterile medium (2YT or
LB) to the centrifuge tube , mix
well, and recover at 37℃ and 200rpm for 60 minutes.
      d. Centrifuge at 5000 rpm for 1 minute to collect the
bacteria, and take about 100 μL of the
supernatant to resuspend the bacteria by gently pipetting and spread on the 2YT or LB medium
containing the corresponding antibiotics.
      e. Invert the plate and incubate overnight in a 37℃
incubator.
![](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-experiment/experiment5.png)

Precautions
      1. Competent cells are best thawed slowly in ice. Add
the target DNA within 8 minutes of
inserting it into ice. Do not keep it in ice for too long, as long-term storage will reduce
the transformation efficiency.
      2. Be gentle when mixing the target DNA.
      3. Transformation of high concentrations of plasmids or
high-efficiency ligation products
can correspondingly reduce the amount of bacteria that are ultimately used for
plating.

2.7 Identify positive clones
(1) Pick single colonies
①600μL of L B liquid medium to 1.5 ml centrifuge tubes respectively
②Pick a single colony on the medium into a centrifuge tube
③shaking at 37℃ for 1 h on a shaker, the bacterial liquid PCR was performed
![](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-experiment/experiment6.jpg)
(2) Bacterial liquid PCR

| Component | Volume |
| --- | --- |
| 2 XHiefft Tm PCR Master Mix | 7.5μL |
| Bacteria liquid | 5.7μL |
| Forward primer | 0.6μL |
| Reverse primer | 0.6μL |
| ddH2O | 0.6μL |

![](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-experiment/experiment7.png)
![](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-experiment/experiment8.jpg)

15μL system(34x)

| Temperature (℃) | Time |
| --- | --- |
| 95 | 3min |
| 95 | 30s |
| 55 | 30s |
| 72 | 1kb/1 min |
| 72 | 10min |
| 4 | ∞ |

2.8 Plasmid Extraction
      1.Culture for 12h until OD600 or so.
      2.Centrifuge leveling, 7800r 5min at room temperature,
supernatant removal.
      3.Add Resuspension Solution 250μL and mix well by
pipetting, transfer to 1.5mL centrifuge
tube.
      4.Add 250μL of Lysis Solution and digest, mix well.
      5.Add 350μL Neutralization Solution and mix well
(13000r, 5min)
      6.Take about 800μL of supernatant and add it to the
special plasmid centrifuge tube (13000r,
5min)
      7.Remove the waste solution, add 500μL Wash Solution
(13000r,1min) and repeat 2 times
      8.13000r,1min centrifugal
      9.Put the special centrifuge column into an empty 1.5mL
centrifuge tube, add 35μL Elution
(preheated at 65℃), cover, and after 2min at room temperature, 13000r, 1min
      10.Store in -20℃ refrigerator
![](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-experiment/experiment9.jpg)
2.9 Enzymatic digestion
1.Essential digestion (digestion system 20μL)
①Endonuclease I (1μL)
②Endonuclease II (1 μL )
③FD Buffer (green) (2μL)
④1301/plasmid containing the target fragment 1000/plasmid concentration
⑤ddH2O Complement
2.PCR setup (20μL system)

| Temperature (℃) | Time |
| --- | --- |
| 37℃ | 25min |
| 65℃ | 20min |
| 4℃ | ∞ |

2.10 Shaking bacteria
    LB liquid medium into the ultra clean bench, ultraviolet 20min, each
50ml centrifuge tube
add 20mL of LB liquid medium and 20μL of AMP, mark well and pour all the bacteria into the
corresponding large tube, if the pour is not exhausted, use the gun to inhale the large
tube, after that put into the shaking E. coli shaker (37℃) for 1 day.

## pGW3 vector construction

3.1 Target primer annealing
①Material
target primer
0.5×TE ( pH8.0 ) 40 μL
②Method
Dissolve the target primers in water into a 10 μM stock solution, add 5 μl of each to 40 μl
of 0.5 × TE (pH 8.0), the final concentration is 1μM; heat at 98°C for 3 min in the PCR
machine, immediately take out the PCR tube, cool to room temperature naturally, and use for
connection.

3.2 Attachment: Preparation of TE
      (1) Preparation of 50ml of 1 M Tris- HCl (pH8.0)
Weigh 6.06 g of Tris base , add 40 ml of ultrapure water to dissolve, add about 2.1 ml of
concentrated HCl dropwise to adjust the pH to 8.0 , and dilute to 50 mL.
      (2) 0.5M EDTA ( pH8.0 ) 50 ml preparation
Weigh 9.306 g of EDTA-Na2·2H2O , add 35 mL of ultrapure water , stir vigorously, adjust the
pH to 8.0 with about 1 g of NaOH particles , and dilute to 50 ml EDTA (The sodium salt will
dissolve only when NaOH is added to adjust the pH to close to 8.0 )
      (3) 1×TE ( 10 m MT ris- HC l , 1mM EDTA )
preparation

| Component | Volume |
| --- | --- |
| 1 MT ris -HC l | 1ml |
| 0.5M EDTA | 0.2 mL |
| Ultra-pure water | Make up to 100 ml |

(4) Preparation of 0.5 × TE

| Component | Volume |
| --- | --- |
| 1 MT ris -HC l | 100 μL |
| 0.5M EDTA | 20μL |
| Ultra-pure waterr | Make up to 20 mL |

Adjust pH to 8.0 with NaOH solution

3.3 The pGW3 plasmid was digested with BsaI
①Material

| Component | Volume |
| --- | --- |
| BsaI enzyme | 1μL |
| plasmid | 1μg |
| 10× NEB buffer | 5μL |
| water | Make up to 50 μL |

②Method
Take 1μg pGW 3 plasmid, digest it with 10 U BsaI-HF at 37℃ for 60 min in a 50 μL reaction ,
use a DNA purification kit, aliquot, and store it in a freezer (reusable).

| Component | Volume |
| --- | --- |
| BsaI enzyme | 1μL |
| plasmid | 1μg |
| 10× NEB buffer | 5μL |
| water | Make up to 50 μL |
| at 37 ℃ for 1h | |

Stop by adding 10 μL of 6× loading buffer to the 50 μL reaction. After enzyme digestion, the
gel was run and the gel was recovered.

3.4 Target ligation reaction
①Material

| Component | Volume |
| --- | --- |
| Material | concentration |
| 10 × T4 DNA ligase buffer | 2 μL |
| The digested pGW3 vector | About 40 ng (use the measured concentration to calculate the amount of sample added) |
| Annealed target primer | 1 μL |
| T4 DNA ligase 40 U | 0.2μL (Takara ligase 350U/ μL add about 0.11μL , add at the end) |
| ddH2O | to 10 μL |

②Method
Connect at room temperature (20 -25℃) for 30 min.

3.5 Convert
①Material
Ligation product 5 μL
Escherichia coli DH5α competent 50 μL
1 bottle of LB solid medium
1 bottle of LB liquid medium
Kana kana :LB solid medium = 1:1 000 ratio
②Method
      a. Take out the DH5a competent cells from -80°C and
insert them into ice quickly. After 5
minutes, wait for the bacterial block to melt, add the target DNA (plasmid or ligation
product) and mix gently by hand to the bottom of the EP tube to avoid using it. Suck it with
a gun and let it sit in ice for 25 minutes.
      b. Heat shock in a 42°C water bath for 45 seconds,
quickly put it back on ice and let it
stand for 2 minutes, shaking will reduce the transformation efficiency.
      c. Add 700ul L B liquid medium without antibiotics to
the centrifuge tube, shake at 37 ℃ for
1h
      d. Centrifuge at 5000rpm for 1 minute to collect the
bacteria, and take about 100ul of the
supernatant to resuspend the bacteria by gently pipetting and spread it on the LB solid
medium containing kanamycin antibiotic .
      e. Place the plate upside down in a 37°C incubator
overnight for 10-12 h .

## Measurement of GABA content

4.1 Sample preparation.
     Weigh about 0.1g of tissue sample into the mortar, add 1mL of
extraction solution,
homogenize on ice, centrifuge at 12000rpm, 4℃ or room temperature for 10min, and take the
supernatant for measurement.
![](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-experiment/experiment10.jpg)
![](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-experiment/experiment11.jpg)
4.2 Detection on the machine
①Preheat the visible spectrophotometer for 30 min, adjust the wavelength to 645 nm, and zero
it with distilled water.
②Thaw all reagents to room temperature (25℃).
③Add to the EP tube in order.

| Reagent name ( μL ) | Assay tube | control tube |
| --- | --- | --- |
| sample | 1 00 | 1 00 |
| Reagent one | 6 0 | 6 60 |
| Reagent two | 2 00 |  |
| Reagent two | 4 00 |  |
| Mix well, take a boiling water bath (95-100℃) for 10min, ice bath to room temperature, showing blue-green color, if it is turbid, centrifuge at 12000rpm for 5min, take all the clear supernatantand transfer it to a 1mL glass cuvette ( optical path 1cm), Read the A value of each tube at 645nm, △A=A determination-A control (each sample needs to be a self-control). | | |

【Note】
1. If the A value of the measurement tube is greater than 1, the sample should be diluted
(diluted with distilled water), and the dilution factor D should be recalculated by
substituting the calculation formula.
If the difference between A measurement and A control hovers around zero, increase the
sample size (e.g. to 0.2g) and recalculate the sample mass W by substituting the
formula.

4.3 Calculation of results
①Standard curve: y = 0.0086x - 0.0033, x is the mass of the standard (μg), y is the
absorbance value ΔA.
![](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-experiment/experiment12.png)
②Calculated by sample mass:
GABA content (μg/g weight) = [(ΔA+0.0033) ÷ 0.0086] ÷ (W×V1÷V) × D
= 1162.8 × (ΔA + 0.0033) ÷ W × D
V-extraction volume, 1 mL; D-dilution, undiluted is 1; W- sample sampling quality

## Agrobacterium infestation(transient transformation for 6d)

      1. Resuscitate Agrobacterium first LB+Kan+Rif plate at
-80°C by scratching and place in dark
culture at 28°C for 48h
      2. Pick single clone, inoculate it in LB liquid medium
containing Kan+Rif, 28℃, 48h,
200r
      3. Centrifuge the bacterial solution at 4500rpm for
10min, remove the supernatant to collect
the bacterium, add MS (i.e. 30g/L sucrose, 4.43g/L MS, PH5.8, 10mmol/LMgCl2, 200umol/L AS
(not high pressure) to resuspend the bacterium, OD adjusted to 0.6-0.9.
      4. The healings cultured for 10-15 d were added to
Agrobacterium infestation solution and
infested for 30 min with shaking and mixing every 10 min during the period.
      5. Filter the longan healing tissue material at the end
of the infestation, blot it slightly
and inoculate it in co-culture medium (normal plus 2,4D medium + 1:1000 cephalosporins, PH
5.8). wash the bacteria after 5-6 d, you can leave some for Gus staining. 1301 empty should
be colorless (positive control); 1301 + target gene (stained), Agrobacterium (no 1301,
colorless ).
      6. Transfer the co-cultured longan healings to a well
pressurized 50 ml centrifuge tube in
an ultra clean bench, rinse with MS medium or water after pressure, and finally rinse with
sterile water + cephalosporin, let stand and shake gently.
      7. Filter and aspirate the water on the surface (filter
paper tweezers should be
high-pressure) received into normal 2,4D + 1:1000 cephalosporin medium, 20d succession.
Attachment.
      1. Cephalosporin: 0.3g+1ml high pressure water to 1.5ml
centrifuge tube, filter and remove
bacteria (green filter, filter once), ready to use
      2. AS: 0.02g acetosyringone (molecular room 4 ℃
refrigerator) + 500ul dimethyl sulfoxide
(drug room under the cabinet) to 1.5ml centrifuge tube, put -20 ℃ can be reused.
      3. Step 3 specific practices.
1:1000 add magnesium chloride to MS suspension, shake well and add 1mL to 50mL centrifuge
tube to aspirate and mix, then pour 25ml of MS suspension, take some liquid to measure OD
value, use the previous MS suspension to adjust the zero. If the OD value is too high,
continue to add MS suspension diluted to OD 0.8 or so. 1:1000 add AS to the already adjusted
OD in the bacterial solution. Put the bacterial solution into the 28 ℃ shaker for half an
hour, during which the table continues to turn on the UV.
      4. Step 4 specific practice.
Strain the material through filter paper until loose, then add the material to the bacterial
solution, add the material to about 20ml. Put it into the shaker of the culture room for
30min.
      5. Step 5.
After shaking, filter with filter paper folded into a funnel shape. After filtering, receive
the co-culture medium and put a clump in the middle.

{% endblock %}

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

![results](https://static.igem.wiki/teams/4113/wiki/wiki-image/background-image/result.png)

↓

![](https://static.igem.wiki/teams/4113/wiki/wiki-image/animation/a4.gif)

Result

Module1：Screening and identification of genes

# Determination of the pathway:

*I*n plants, there are two pathways for GABA synthesis
and conversion: one is the
synthesis of
GABA from Glutamic acid catalyzed by glutamic acid decarboxylase (GAD), called the GABA
shunt; the other is the conversion of polyamine degradation products to form GABA, called
the polyamine degradation pathway.
  *I*n higher plants, the metabolism of GABA is mainly
accomplished by three
enzymes, firstly,
the irreversible decarboxylation of L-glutamic acid (Glu) at the α-position under the action
of GAD to form GABA, and then the reaction of GABA with pyruvate and α-ketoglutarate
catalyzed by GABA transaminase (GABA-T) to form succinate. Then, GABA reacts with pyruvate
and α-ketoglutarate to form succinic semialdehyde, and finally succinic semialdehyde
dehydrogenase (SSADH) catalyzes the oxidative dehydrogenation of succinic semialdehyde to
form succinic acid, which finally enters the tricarboxylic acid cycle (krebs circle). This
metabolic pathway constitutes a branch of the TCA cycle, called the GABA branch.
![result1](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result1.jpg)

*I*n plants, GAD presents in the cytoplasm and GABA-T
and SSADH in the mitochondria
together
regulate the metabolism of the GABA branch, where GAD is the rate-limiting enzyme for GABA
synthesis. Plant GAD contains a calmodulin (CaM) binding region, and GAD activity is not
only co-regulated by Ca2+ and H+ concentrations, but also influenced by the
concentration of
the GAD coenzyme, pyridoxal phosphate (PLP), and the substrate glutamate. This dual
regulatory mechanism links the cellular accumulation of GABA to the nature and severity of
environmental stresses. Cold stress, heat stress, osmotic stress and mechanical injury all
increase the Ca2+ concentration in the cytosol, and
Ca2+ binds to CaM to form the Ca2+/CaM
complex, which is capable of stimulating GAD gene expression and increasing GAD activity
under normal physiological pH conditions; whereas acidic pH stimulates the appearance of GAD
as a result of stress lowering the pH of the cell and slowing down the acidic harm to the
cell. The GABA branch in plants is considered to be the main pathway for GABA synthesis.
Currently, most studies have focused on how to increase GAD activity to achieve GABA
enrichment.
  *T*herefore, we chose the pathway of GABA generation
from glutamate catalyzed by GAD in the
GABA branch.
![result2](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result2.png)

# GAD and CaM gene screening:

*B*y using the CaM gene of Arabidopsis as the search
object, we used the BLAST
function of
TBtools to compare the CaM genes in longan in the three-generation genome database of
longan, kept all the IDs of the screened CaM, extracted the corresponding protein sequences
using the Fasta Extract function of TBtools, and constructed the CaM genes in Arabidopsis
and rice by MEGA7.0 We found that only two DlCaM genes were evolutionarily related to
Arabidopsis and rice, and identified these two genes as the CaM genes we were looking for.
Structural domain validation was performed to screen out sequences without CaM structural
domains and EF-hand, and the resulting candidate protein IDs were used to obtain the
protein, CDS and gene sequences of the CaM gene family by the sequence extraction tool of
TBtools (Fasta Extract). Longan genome sequences, protein sequences, gDNA sequences and gff
files were derived from our laboratory database.
![result3](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result3.png)
  *T*he conserved structural domain analysis showed that
all CaM gene family members contained
the PTZ00184 structural domain, further supporting the correctness of the two selected
longan CaM genes.
![result4](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result4.png)
  *B*oth genes were used in the gene editing CRISPR-Cas9
knockdown experiments based on the
expression profiles showing that both CaMs in Longan were highly expressed at all three
stages of somatic embryogenesis.
![result5](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result5.png)

# GAD gene screening:

*T*he GAD gene of Arabidopsis was used as the search
sequence, and the BLAST
function of
TBtools was used for comparison analysis, and the sequences with sequence similarity higher
than 70% were retained, and the structural domain was verified by NCBI CDD search
(https://www.ncbi.nlm.nih.gov/cdd) to screen out the sequences that did not contain the
Glu-decarb-GAD protein structural domain, and finally four DlGAD genes were obtained. Among
them, GAD2 is special, with a structural domain different from all other longan GAD genes.
![result6](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result6.png)
  *T*ranscriptome analysis of six GAD genes revealed
that the expression of GAD2 and GAD5 was
high and GAD5 was differentially expressed, so these two genes were used for the
construction of overexpression vectors.
![result7](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result7.png)

Module2：Construction of GAD overexpression vector and CaM knockdown
experiments

  Through a series of experiments, we successfully constructed an overexpression
vector for GAD
and a pGW3 vector
(Source: The New Zealand Institute for Plant and Food Research
Limited) for knocking out CaM.

# Construction of GAD overexpression vector

*1.1 PCR amplification*
*①Primer*

| ID Rename | F/R | Primer | Tm | Enzyme cut site |
| --- | --- | --- | --- | --- |
| GAD2 | F | GGGGTACCATGGCTCAGACCGTTGCATATG | 62.4 | KpnⅠ |
| R | ACGCGTCGACTCACTTATGGAGACCACAAGCACA | 62.2 | SalⅠ |
| GAD5 | F | CGGGGTACCATGGTGATATCTTCAACAACAGCG | 61.1 | KpnⅠ |
| R | AACTGCAGTTAGCATGCTCCGGCTCTCC | 62.7 | PstⅠ |

*②Electrophoresis results*
*DlGAD2*
![result8](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result8.png)
 Figure 1：PCR amplification product of DlGAD2
The band marked in red is the band run for GAD2, and the length of GAD2 is 1428bp.

*DlGAD5*
![result9](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result9.png)
Figure 2 PCR amplification product of DlGAD5
As the figure is marked with red box is the band run out for GAD5, the length of GAD5 is
1473bp.
*1.2 Fungal plate growth*
The following pictures are all the colonies that grew after the plate was coated.
![result10](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result10.png)
Figure 3 Bacteria plate
*1.3 Colony PCR results*
![result19](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result19.png)
Figure 4 DlGAD2 bacteriophage PCR of Pcombia1301SN-DlGAD2
vector
![result11](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result11.png)
Figure 5 DlGAD5 bacteriophage PCR of Pcombia1301SN-DlGAD2
vector
So far, after sending the test to the biological company, we successfully cloned DlGAD2 and
DlGAD5.
*④Dual enzyme digestion*
![result12](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result12.png)
Figure 6 Enzyme-digested product

# CRISPR/Cas9 knockdown DlCaM experiments

*2.1 CaM knockdown experiments*
*①Plasmids*
   The vector we used in our experiments was Genovo-W-3, commonly referred to
as pGW3 (Source: The New Zealand Institute for Plant and Food
Research Limited)
. pGW3 has
a fragment length of 16,654 bp. We cleaved it with BsaⅠ and gum recovered a fragment of size
15,429 bp, the pGW3 backbone. We used T4 ligase to ligate the sgRNA to the pGW3 backbone to
obtain the recombinant plasmid.
![result13](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result13.png)
Figure7: Genovo-W-3 plasmid mapping
*②Enzyme digestion*
   Plasmid pGW3 was digested with Bsa I:
![result14](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result14.png)
Figure 8 Enzyme-digested product
*③Transform*
   The ligation product was transformed into E. coli DH5α and coated on
medium with kana.
![result15](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result15.png)
Figure9: Transformed colonies of pGW3-ΔDlCaM1 recombinant
![result16](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result16.png)
Figure10: Transformed colonies of pGW3-ΔDlCaM2 recombinant
vector
*④Identification of positive clones*
After 10 -12h, single clones were selected for colony PCR identification and sequencing to
confirm whether the target was attached to the vector. The bacteriophage with correct PCR
bands will be selected and a tube of large shake extracted plasmids will be sequenced.
![result17](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result17.png)
Figure 11 Bacteriophage PCR result
*2.2 Transformation and Infestation*
①Transfer of recombinant plasmid into Agrobacterium
![result18](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result18.png)
Figure12: pGW3-ΔDlCaM1 recombinant vector and pGW3-ΔDlCaM2
recombinant vector Agrobacterium bacteriophage PCR
②Infestation
   Agrobacterium tumefaciens carrying the recombinant plasmid was infiltrated
into longan
healing tissues and transiently transformed for 6 d. Our team is currently working on
verifying the successful transfer of the recombinant plasmid into longan and will next test
the GABA content in the gene edited longan healing tissues.

Module3：Exogenous processing results

   We completed experiments on the effects of transient high temperature
stress and blue light
treatment on the endogenous GABA content of Longan somatic embryos.
   We placed longan healing tissues in good growth condition (spherical
embryo period) in a
thermostat and a blue light incubator for adversity treatment, the temperature of the
thermostat was adjusted to 40°C and the parameters of the blue light incubator were adjusted
to 32umol-m-2-S-2, and the samples were collected every 5 min until 120 min, and the GABA
content in the samples was measured according to the procedure of the purchased GABA kit.
The following model was constructed based on the measured data, and the obtained data were
analyzed by SPSS to obtain the results of significance level.
![result20](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result20.png)
Blue light processing
![result21](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result21.png)
High temperature treatment
   GABA is a kind of adversity protein, under the condition of adversity
stress (blue light and
high temperature stress), the content of GABA will first show an increasing trend, but this
increasing trend does not last all the time, reaching a certain limit, this increasing trend
begins to gradually change to a decreasing trend. Under blue light treatment conditions, the
GABA content reached its peak at about 30 min of treatment, while under high temperature
treatment conditions, the GABA content reached its peak at 45 min of treatment. We can rely
on this conclusion to guide the production of high-yielding GABA cell factories by
subjecting longan healing tissues to 30 min of blue light treatment and 45 min of high
temperature treatment to increase the GABA content as a way to achieve high yield.
![result22](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result22.png)
![result23](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result23.png)
High temperature treatment
![result24](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result24.png)
significance level (P=0.05)
![result25](https://static.igem.wiki/teams/4113/wiki/wiki-image/lab-result/result25.png)
Blue light treatment significance level (P=0.05)
   According to the results given by SPSS software, the sample variance was
significant at
P=0.05, under the condition of blue light treatment, 0.035 <0.05, indicating that the
variance between samples was chi-square, which also indicates that blue light treatment
had a significant effect on GABA content in longan healing tissue; at P=0.05, under the
condition of high temperature treatment, the sample variance was significant at 0.037 <
0.05, indicating that the variance between samples was chi-square, which could also
indicate that high temperature treatment also had a significant effect on GABA content
in longan healing tissues.

{% endblock %}