iGEM 2022 — Team canton-hs

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{% block title %}Notebook{% endblock %}
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Notebook

Day 1 (2022. 8.3)

1. Laboratory safety training
2. Configure LB medium
3. PCR amplification of tnaC and amilGFP template DNA
4. Inoculate pTrc99K and pET28a-inaK-gldh containing strains
5. gel extraction

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-notebook01.jpg)

Day 2 (2022. 8.4)

1. Plasmid pTrc99K and pET28a-inaK-gldh were extracted
2. SLC7A5 gene fragments amplification
3. Gel electrophoresis
4. Gel extraction of tnaC-amilGFP, SLC7A5 gene fragments
5. Double-enzyme digestion of Overlap's large segment
6. extraction of the digested fragments
7. ligate the DNA fragments by T4 DNA ligase and transform them into Top10 competent cells

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-notebook02.jpg)

Day 3 (2022. 8.5)

1. Transformants were identified by colony PCR
2. Select monoclonal and culture it in liquid LB medium

Day 4 (2022. 8.6)

1. Plasmids extraction and verification by double-enzyme digestion
2. Send the correct plasmids to the company for Sanger sequencing

Day 5（2022. 8.7）

1. Transform the pET28a-inaK-gldh plasmid to BL21 (DE3) and incubate at 37℃.
2. Inoculate the pTrc99k-tnaC-amilGFP transformants in LB(Amp+) liquid medium overnight at 37℃, 220rpm.

Day 6 (2022. 8.8)

1. Inoculate the pET28a-inaK-gldh plasmid containing BL21 (DE3) monoclonal in 10 mL LB(Kan+) liquid medium and
culture at 37℃, 220rpm for 12h.
2. Transfer the cultured medium of pTrc99k-tnaC-amilGFP into several bottoms of LB medium with different
concentrations of tryptophan, and take 200μL samples at different times.

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-notebook03.jpg)

Day 7 (2022. 8.9)

1. Transfer the cultured medium of pET28a-inaK-gldh into fresh LB(Kan+) liquid medium and incubate at 37℃, 220rpm
until OD600 is around 0.6, add IPTG to induce protein expression.
2. Measure the fluorescence of pTrc99k-tnaC-amilGFP samples.
3. Purification of the inaK-gldh protein through the Ni-NTA method.
4. Prepare SDS-PAGE protein gel and protein expression detection.

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-notebook04.jpg)

Day 8 (2022. 8.10)

1. Glutamatase activity detection
2. Analyze and organize data

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-notebook05.jpg)

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{% block title %}Engineering Success{% endblock %}
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![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-banner.jpg)
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Engineering

Introduction

Various neurotransmitters in the brain are closely related to amino acid metabolism in food. The glutamate is the
precursor of γ-aminobutyric acid, and γ-aminobutyrate expression level can affect ASD symptoms such as sleep
disorders and affective disorders. Studies have found that the blood levels of glutamate rise significantly in
ASD.

This project develops a glutamate dehydrogenase activity detection system to verify if it could be used to diagnose
ASD. In this project, we constructed an inak-gldh expression plasmid and transformed it into BL21(DE3) competent
cells. We detected the expression level of inak-gldh by SDS-PAGE, and measured the enzyme activity by adding
L-glutamate to the reaction system. Our experimental data indicated its significant effect on glutamic acid
dehydrogenation. Therefore, our product will provide an efficient ASD-detecting biosensor.

How we design our plasmid

Because the T7 promoter and T7 RNA polymerase have strong ability in translation and usually be used as protein
expression, we chose pET28a-vector and E. coli BL21(DE3), with T7 promoter and T7 RNA polymerase respectively, to
express our target protein inak-gldh. To achieve this, we optimized the DNA sequences of inak-gldh and inserted them
into the HindIII and NcoI sites of the pET28a vector (Figure 1.), and transformed the recombinant plasmid into E.
coli BL21(DE3) for protein expression.

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-engineering01.jpg)
Figure 1. The map of recombinant plasmid pET28a- inak-gldh.

How we build our plasmid

To build the plasmid, we let the synthetic company synthesize the DNA fragment of optimized inak-gldh and inserted
it into the HindIII and NcoI sites of the pET28a vector (Figure 2A). Then, we send it to the company for Sanger
sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure
2B), and the plasmid pET28a- inak-gldh was successfully constructed. And the last step was extracting the
recombinant plasmid and transforming it into E. coli BL21(DE3) competent cells, so that can be used to express the
inak-gldh proteins.

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-engineering02.jpg)

Figure 2. The result of recombinant plasmid pET28a- inak-gldh.
A. double-enzyme digestion of the recombinant plasmid, 1: the plasmid pET28a, 2: the plasmid pET28a- inak-gldh, B.
The sequencing blast results of the recombinant plasmid

How we test our glutamate dehydrogenase inak-gldh

a) Protein expression verification

In order to verify the inak-gldh protein expression level, we cultured pET28a- inak-gldh containing BL21(DE3) strain
in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.6. After overnight induction
and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins.
Next, we used the SDS-PAGE method to verify the expression level of the target protein (Figure 3). As a result, we
could significantly find the band at the correct size.

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-engineering03.jpg)
Figure 3. SDS-PAGE detection of inak-gldh protein expression.

b) Measure the standard curve of NADPH

Glutamate dehydrogenase could use L-glutamate as a substrate, with reversible oxidation and deamination under the
action of coenzyme (NAD + or NADP +), and the NADH or NADPH generated by the reaction has an obvious absorption peak
at 340 nm. To detect glutamate dehydrogenase activity, the amount of NADPH generated by the hydrogenase catalytic
reaction was measured spectrophotometrically. Absorption values at 340 nm were determined using a UV-visible
spectrophotometer (Figure 4). One unit of enzymatic activity was defined as the production of 1 μ mol of the reduced
product, NADPH, per OD600 cells per minute.

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-engineering04.jpg)

Figure 4. the reaction principle of glutamate dehydrogenase

NADPH was diluted to 10 μM, 50 μM, 100 μM, 400 μM, 500 μM, and 500 μM, and the absorption peaks at 340nm at each
concentration were measured by spectrophotometer. With the light absorption value of NADPH at 340 nm as the vertical
coordinate and the corresponding concentration of NADPH (μM) as the abscissa, the linear fitting was performed to
obtain the linear regression equation corresponding to the standard curve and the standard curve.

c) Functional test of inaK-gldh

Cells containing the expression vector pET28a-inaK-gldh were centrifuged and washed twice with 100mM Tris-HCl (pH
8.0) buffer after induction. The standard reaction system contained bacterial cells (OD600-1.0), 100mM Tris-HC1
buffer (pH 8.0), sodium L-glutamate (final concentration 2mM), and NADP + (final concentration 0.5mM). The reaction
was performed at 60°C in a 1.5mL centrifuge tube for 2min and was terminated by centrifugation of the bacteria at
12,000 rpm for 1min. The absorptive values at 340 nm were measured using a UV-visible spectrophotometer. As a
result, the enzyme activity of glutamine dehydrogenase is around 567.92975 U/mL. So that the biosensor we
constructed worked well.

The enzyme activity calculation formula is as follows

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-engineering05.jpg)

| △T | △A | GDH（U/mL） |
| --- | --- | --- |
| 120.0900 | 0.3533 | 567.92975 |

\* Vsample: 0.05 mL, V total: 1×10-3 L, ε: NADH molar extinction coefficient, 6.22×103
L/mol/cm, d:
Cuvette light
diameter, 1 cm, ∆T: Reaction time, 2 min.

How we learn from our project

We have already collected the figures from our experiments. Glutamate dehydrogenase plays an important role in
L-glutamate metabolism. After transforming the sequence-optimized gene inaK-gldh into the host strain, we can easily
detect the metabolites NADPH using our biosensor. What’s more, we measured the activity of inaK-gldh, and found that
the inaK-gldh worked well in the E. coli system.

The task of developing new portable tests and effective treatments is imminent. In the future, when the biosensor is
improved, it may be applied to ASD detection, which can provide a new tool for disease diagnosis, and the earlier
the diagnosis is made, the better the outcome.

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{% block title %}Experiments{% endblock %}
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Experiments

Bacterial culture

Making LB Medium

What we needed was 100mL of solid medium and 350mL of liquid medium. According to the formula, every 1L of medium
requires 10g of tryptone, 5g of yeast extract, 10g of NaCl, and ddH2O. For the solid medium, a 1% concentration of
agarose powder is needed for solidification.
After adding the ingredients together, we used a plastic sheet with filter paper in the middle to cover the flask
and filter possible impurities during autoclaved sterilization. We sterilized for about 20 minutes and let the
medium cool down. For the solid medium, we added 50µL of Kanamycin or Ampicillin antibiotic. And for the final step,
we poured the melted solid medium into the Petri dishes on the clean bench.

Construction of plasmids

Overview:

1. PCR
2. PCR Gel Electrophoresis
3. PCR Gel Purification
4. Double-enzyme digestion
5. T4 DNA ligase
6. DNA transformation to competent cells

1. PCR:

1) Use a pipette to add reagents in a PCR tube and mix up (which contains enzymes required for PCR; dNTPs; ddH2O;
and Buffer). Next, add the primers into the tube. Afterward, divide the prepared mixture into 2 PCR tubes.
2) Put the PCR tube into the PCR machine. Close the lid and set the program.

| Steps | Temperature | Time | Number of cycles |
| --- | --- | --- | --- |
| Initial denaturation | 94 ℃ | 5 min |  |
| Denaturation | 94 ℃ | 30 sec | 33 |
| Primer annealing | 55 ℃ | 45 sec |
| Extension | 72 ℃ | 2 min |
| IFinal extension | 72 ℃ | 5 min |  |

2. PCR Gel Electrophoresis

1) Since the DNA contains a negative electric charge, we placed the gel with wells toward the negative charge of
the buffer tank, as the other side is positively charged. Keep in mind that the TAE buffer needs to fully cover
the gel.
2) Added DNA loading buffer to the PCR product, according to its concentration.
3) After the electrophoresis, we put the gel under the ultra slim LED illuminator to see the DNA and cut out the
gel with DNA fragments for the next step.

3. PCR Gel Purification

1) Cut down the gel containing the target fragment and calculate the weight difference in the EP tube from adding
the gel by weighing the tube before and after adding the gel
2) Add 500µL B2 buffer to the gel, and water bath at 50℃ for 5-10 minutes to make the gel melt in the buffer
3) Move all the liquid into the absorption column with membrane, centrifuge for 30s, at 8000×g
4) Add 500µL Wash Solution, 9000×g centrifuge for 30s, repeat this step again
5) Centrifuge again for 2 minutes, 12000×g.
6) Put the absorption column into a clean 1.5mL centrifuge tube, add 30µL Elution Buffer, put it at room
temperature for 2 minutes, and centrifuge for 1 minute to keep the DNA solution.
7) Measure the DNA concentration using the Nanodrop machine.

4. Double-enzyme digestion

1) add 2 μL each of the restriction enzyme into one micro-centrifuge tube.
2) add 5μL buffer, 10μL ddH₂O, and 1μL DNA into each micro-centrifuge tube.
3) 37℃ incubation for 1h.
4) Agarose Gel Electrophoresis and gel extraction.

5. T4 DNA ligase

1) add 1 μL T4 DNA ligase, 1 μL T4 DNA ligase buffer, 1 μL DNA fragment, 1 μL plasmid vector, and 5 μL ddH₂O into
the microcentrifuge tube.
2) 22 ℃ water bath for 30 minutes.

6. DNA transformation to plasmid

1) Put the chemically competent cells DH5α on ice for 1-2 minutes.
2) Mix the DH5α competent cells with precooled recombinant plasmids. Ice bath for 30 minutes.
3) Heat shock at 42°C for 1 minute and put on ice immediately for 2 minutes.
4) Add 700μL LB medium and incubate it at 37℃, 200rpm for 60 minutes.
5) Centrifuge 8000rpm 1 min, get rid of 650µL of supernatant.
6) Resuspend the E. coli and coat it on the solid medium.

Identification of recombinant plasmids

Overview:

1. Colony PCR
2. Plasmid Extraction

1. Colony PCR

ystem:
1. We added 5µL ddH₂O .
2. Added 10µL Primer S. Mixer (which includes dNTP, DNA polymerase, and reaction buffer).
3. Added 3µL of DNA template.
4. Added 1µL each of forward and reverse primer.
program:
1) 95℃, 5min, pre-degenerate
2) 95℃, 10s, DNA degeneration
3) 58℃, 30s, annealing
4) 72℃, elongation
5) 72℃, 10min, fully elongate
6) 4℃, infinite time, preserve

2. Plasmid Extraction

We cultured E. coli that to do plasmid extraction
1) Inoculate the bacteria and culture overnight, and centrifuge the cultured medium at 12000rpm for 1 min. Discard
the supernatant
2) Add 500μl of the balance solution to the adsorption column, centrifuge the discard the filtrate
3) Add 250μl of Buffer SP1 from the Diamond Mini Plasmid Kit to the pellet cells. Add 250μl Buffer SP2 and 350μl
Buffer SP3 and mix well.
4) Centrifuge at 12000rpm for 10 minutes, add the supernatant to the adsorption column, and centrifuge at 8000rpm
for 30s.
5) Add 500 mL of wash solution, centrifuge at 9000×g for 30 seconds, and pour off the liquid in the tube (repeat
once)
6) Centrifuge the empty adsorption column at 9000 ×g for 1 minute
7) Put the adsorption column into a new 1.5mL centrifuge tube, add 100 mL of Elution Buffer to the adsorption
membrane, stand at room temperature for 1 minute, and centrifuge for 1 minute.

Protein expression

1) Inoculate the plasmid-containing strain in LB medium with corresponding antibiotics and culture overnight at 37℃,
200-220rpm.
2) Transfer the cultured medium into a 500mL LB culture medium, and add IPTG when the OD600 is around 0.6.
3) After overnight culturing, we verified the protein expression level by SDS-PAGE.

Tryptophan detection

1) Inoculate the single colonies of the engineered strains in a triangular flask containing 10mL of fresh LB medium
(containing antibiotics), and incubated at 37°C, 220 rpm for 12h.
2) 1 mL of bacterial cultured medium was added to a triangular flask containing 100mL of fresh LB medium (including
100mg/L Ampicillin) and incubated at 37°C, 220 rpm until the 0D600 was around 0.6.
3) The solution was divided into conical flasks, adding 50ml of cultured medium and L-tryptophan to a final
concentration of 0.75mmol/L, 1mmol/L, 1.5mmol/L, 2mmol/L, 2.5mmol/L, and 3mmol / L and cultured at 22°C, 220 rpm for
7 hours. Samples were taken at 1h, 2h, 4h, 5h, 6h, 7h.
4) Fluorescence intensity was detected immediately by using 200 μL bacterial solution samples to a 96-well black
microplate plate

Glutamine dehydrogenase activity measurement assay

1) We used the Glutamate Dehydrogenase (GDH) Activity Assay Kit to measure the protein activity.
2) The bacteria cells were collected by centrifugation into centrifugal tubes, treated samples in the proportion of
bacteria: extract volume (mL) (500-1000): 1 (1 mL of 5 million bacteria), crushed by ice bath sonicated (power 20%
or 200 W, ultrasonic for 3s, 10s interval, 30 repeats), centrifuged at 4℃ 8000 g for 10 min, and the supernatant was
placed on ice for testing.
3) Absorption values at 340 nm were determined using a UV-visible spectrophotometer.

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{% block title %}Results{% endblock %}
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Results

1. Construction of the ASD-detection-related plasmids

We design three plasmids: The DNA sequences of the tnaC-amilGFP was inserted into the pTrc99K vector, and the
codon-optimized inaK-gldh and SLC7A5 were inserted into the HindIII and NcoI sites of the pET28a vector,
respectively. These plasmids were shown in Figure1.

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-results01.jpg)

Figure 1. Protein expression plasmids in this project.
A. the plasmid pTrc99K-tnaC-amilGFP,
B. the plasmid pET28a-inaK-gldh,
C. the plasmid pET28a-SLC7A5.

In order to build our plasmids, we let the synthetic company synthesize the codon-optimized inaK-gldh fused DNA
fragment and integrate it into the pET28a vector (Figure 1C).
Next, we amplified the tnaC, amilGFP, and SLC7A5 DNA fragments by PCR. Then, we fused the tnaC and amilGFP through
overlap-PCR (Figure 2). We extracted the target DNA fragments and digested the tnaC-amilGFP and pTrc99K with NcoI
and HindIII, and ligated by T4 DNA ligase. The DNA fragment SLC7A5 and pET28a with EcoRI and XhoI, and ligated by T7
DNA ligase. The recombinant plasmids were transformed into E. coli Top10 competent cells and coated on LB medium
plate with corresponding antibiotics.

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-results02.jpg)

Figure 2. Gel electrophoresis results of target gene fragments.
M: DNA Marker,
A. The gene fragments of tnaC and amilGFP,
B. The gene fragments of SLC7A5,
C. The gene fragments of tnaC-amilGFP.

We verified our recombinant plasmids by colony PCR (Figure 3), and we inoculated the correct strains and extracted
plasmids.

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-results03.jpg)

Figure 3. Colony PCR verification of pET28a-SLC7A5.

We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing
comparison results showed that there were no mutations in the ORF region (Figure 4), and the plasmid was
successfully constructed. So far, we have successfully obtained ASD-detection-related plasmids.

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-results04.jpg)

Figure 4. The sequencing data mapped to the recombinant plasmids.
A. the sequencing data mapped to the plasmid pTrc99K-tnaC-amilGFP,
B. the sequencing data mapped to the plasmid pET28a-inaK-gldh,
C. the sequencing data mapped to the plasmid pET28a-SLC7A5.

2. Protein expression and purification

In order to obtain the inaK-gldh and SLC7A5 proteins, we transformed the recombinant plasmids into E. coli
BL21(DE3), inoculated the recombinants and added different concentrations of IPTG to induce protein expression when
the OD600 reached 0.6. After overnight induce and culture, we collected the cells and ultrasonic
fragmentation of
cells to release the intracellular proteins. Next, we verified the expression level of the target protein by
SDS-PAGE (Figure 5). As a result, the recombinants successfully expressed our target protein.

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-results05.jpg)

Figure 5. SDS-PAGE detection of target proteins.
A. the result of expressed inaK-gldh protein,
B. the result of expressed SLC7A5 protein.
P: precipitation, S: supernatant, T: through, W: wash, E: purified supernatant.

3. Proteins activity test

Overview

To confirm the ability of our ASD-detection-related biosensor, we used E. coli BL21(DE3) as host strains and
detected the related proteins’ activity. There are several markers that could be chosen to detect, such as
tryptophan and glutamic acid.
To better show the activity of our biosensor, we constructed a recombinant plasmid pTrc99k-tnaC-amilGFP as a
reporter of tryptophan detection. We measured the relationship of the fluorescence intensity with the concentration
of tryptophan and the incubation time.
What’s more, we also measured the activity of the glutamine dehydrogenase by constructing the plasmid
pET28a-inaK-gldh and transforming it into BL21(DE3). We purified the protein and developed an in vitro reaction
platform to detect its activity.
The results showed that the biosensor we developed could be used to detect ASD. A detailed analysis of the biosensor
is given below.

a) Tryptophan detection

Single colonies of the engineered strains containing the recombinant plasmid pTrc99K-tnaC-amilGFP were picked,
placed in a triangular flask containing 10mL of fresh LB medium (containing 100mg/L Ampicillin), and incubated at
37°C, 220 rpm for 12h. 1 mL of bacterial cultured medium was added to a triangular flask containing 100mL of fresh
LB medium (including 100mg/L Ampicillin) and incubated at 37°C, 220 rpm until the OD600 was around 0.6.
The solution
was divided into conical flasks, adding 50ml of cultured medium and L-tryptophan to a final concentration of
0.75mmol/L, 1mmol/L, 1.5mmol/L, 2mmol/L, 2.5mmol/L, and 3mmol / L and cultured at 22°C, 220 rpm for 7 hours, and
samples were taken at 1h, 2h, 4h, 5h, 6h, 7h. Fluorescence intensity was detected immediately by using 200 μL
bacterial solution samples to a 96-well black microplate plate (Figure 6).

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-results06.jpg)

Figure 6. the Fluorescence intensity result of L-tryptophan.

b) Glutamine dehydrogenase activity measurement assay

Glutamate dehydrogenase could use L-glutamate as a substrate, with reversible oxidation and deamination under the
action of coenzyme (NAD + or NADP +), and the NADH or NADPH generated by the reaction has an obvious absorption peak
at 340 nm. To detect glutamate dehydrogenase activity, the amount of NADPH generated by the hydrogenase catalytic
reaction was measured spectrophotometrically.
We used the Glutamate Dehydrogenase (GDH) Activity Assay Kit to measure the protein activity. The bacteria cells
were collected by centrifugation into centrifugal tubes, treated samples in the proportion of bacteria: extract
volume (mL) (500-1000): 1 (1 mL of 5 million bacteria), crushed by ice bath sonicated (power 20% or 200 W,
ultrasonic for 3s, 10s interval, 30 repeats), centrifuged at 4℃ 8000 g for 10 min, and the supernatant was placed on
ice for testing. Absorption values at 340 nm were determined using a UV-visible spectrophotometer.
Cells containing the expression vector pET28a-inaK-gldh were centrifuged and washed twice with 100mM Tris-HCl (pH
8.0) buffer after induction. The standard reaction system contained bacterial cells (OD600-1.0), 100mM
Tris-HC1
buffer (pH 8.0), sodium L-glutamate (final concentration 2mM), and NADP + (final concentration 0.5mM). The reaction
was performed at 60°C in a 1.5mL centrifuge tube for 2min and was terminated by centrifugation of the bacteria at
12,000 rpm for 1min. The absorptive values at 340 nm were measured using a UV-visible spectrophotometer. As a
result, the enzyme activity of glutamine dehydrogenase is around 567.92975 U/mL. So that the biosensor we
constructed worked well.

The enzyme activity calculation formula is as follows

![](https://static.igem.wiki/teams/4291/wiki/t-canton-hs-engineering05.jpg)

| △T | △A | GDH（U/mL） |
| --- | --- | --- |
| 120.0900 | 0.3533 | 567.92975 |

\* Vsample: 0.05 mL, V total: 1×10-3 L, ε: NADH molar extinction coefficient, 6.22×103
L/mol/cm, d:
Cuvette light
diameter, 1 cm, ∆T: Reaction time, 2 min.

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