iGEM 2021 — Team Xiamen_City

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# Team:Xiamen City/Notebook

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# Notebook

# 0806

a) Safety training

b) Study basic techniques used during experiments, and the PCR technology

c) Prepare culture medium (YPD medium and LB medium).

d) Transfer Cas9 and gRNA plasmid DNA into two groups of Escherichia coli.

e) Apply PCR to the upstream and downstream homology arms.

![](https://static.igem.org/mediawiki/2021/0/09/T--Xiamen_City--img_notebook_1.jpg)
![](https://static.igem.org/mediawiki/2021/6/69/T--Xiamen_City--img_notebook_2.jpg)

# 0807

f) Learn how to test OD

g) Learn the electrophoresis technology

h) Proliferate the two groups of Escherichia coli.

i) Transform the yeast cells with Cas9 plasmid DNA into competent state.

![](https://static.igem.org/mediawiki/2021/5/55/T--Xiamen_City--img_notebook_3.jpg)

15KM 1 2 3 4

1: pgaA PCR product

2: pgaA PCR product

3: HR-L PCR product

4: HR-R PCR product

# 0808

j) Learn the column extraction method

k) Extract Cas9 and gRNA plasmid DNA from the two groups of Escherichia coli.

![](https://static.igem.org/mediawiki/2021/1/18/T--Xiamen_City--img_notebook_4.jpg)

15KM 1 2 3 4 15KM 5 6 7 8

1: pHCas9-Nourse-1

2: pHCas9-Nourse-2

3: pYES2-gRNA-hyg-MCS-1

4: pYES2-gRNA-hyg-MCS-2

5: HR-L PCR product

6: HR-L PCR Product

# 0809

l) Prepare the double antibiotics YPD medium.

m) Cultivate the yeast with single fungus containing cas9 plasmid

n) Apply PCR to the pgaA sample.

# 0811

o) Apply PCR to integrate the upstream, downstream homology template, and pgaA.

p) Cultivate yeast cells that contains Cas9 plasmid.

q) Rebuild 3 PCR samples (up+pgaA, pgaA+down, up+pgaA+down)

# 0812

r) Examine previous PCR results.

s) Extract homology repair template.

t) Transfer the gRNA plasmid and homology repair template into the yeast competent cell
containing the Cas9 plasmid.

![](https://static.igem.org/mediawiki/2021/2/2e/T--Xiamen_City--img_notebook_5.jpg)

15K 1 2

# 0813

u) Prepare Congo red solution.

v) Prepare NaOH solution

w) Prepare NaCl solution

# 0814

x) Prepare pectin medium for function test

# 0815

y) Test the yeast solution at different concentration

![](https://static.igem.org/mediawiki/2021/a/a4/T--Xiamen_City--img_notebook_6.jpg)

# 0816

z) Extract plasmid and send out for sequencing

# 0817

aa) Sequencing feedback received

![](https://static.igem.org/mediawiki/2021/c/c7/T--Xiamen_City--img_notebook_7.jpg)

# 0917-0923

bb) Induction experiments

cc) DNS Test

![](https://static.igem.org/mediawiki/2021/6/68/T--Xiamen_City--img_notebook_8.jpg)

## Contact Info

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# Team:Xiamen City/Engineering

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# Engineering

# Background

Pectin is a kind of high molecular carbohydrate existing in the cell wall of plants. The main chain
is polymerized by galacturonic acid (PGA), while the side chain is composed of various
polysaccharides such as galactose, rhamnose, and xylose. In fruit wine production, *Saccharomyces
cerevisiae* uses the sugars in fruit juice to ferment to produce alcohol. It is necessary to
add pectinase to destroy the pectin in the cell wall to increase the juice yield and increase the
dissolution of aromatic substances such as pigments or terpenes. In addition, pectinase can also
eliminate the turbidity of fruit wine caused by pectin colloids, so as to ensure the light
transmittance and stability of fruit wine to the greatest extent. Most of the pectinase currently on
the market is a composite enzyme obtained from the fermentation of *Aspergillus*. This
undoubtedly increases the cost of fruit wine production.

There have been successful cases of exogenous expression of pectinase. All these achievements aimed
to obtain high-yielding pectinase production strains, but yeast strains that can both decompose
pectin and complete alcohol fermentation have never been obtained.

# Design

This project uses the CRISPR-Cas technology to realize the heterologous expression of the cohesive
galacturonase gene endo-pgaA of *Aspergillus niger* SC323 in the fruit wine yeast, and obtain a
yeast
strain that can degrade pectin. This will help to reduce the cost of fruit juice and fruit wine
production process.

A double-plasmid expression system of CRISPR-Cas9 was designed to edit genome of *Saccharomyces
cerevisiae* (Figure 1). One plasmid is pHCas9-Nours plasmid expressing Cas protein and another is
pYES2-gRNA-hyg-MCS plasmid containing sgRNA. First, pHCas9-Nours plasmid was introduced yeast cells,
and then the pYES2–gRNA-hyg-MCS plasmid and the homology arm for repair into the wine yeast. After
obtaining the transformant, verify endo-pgaA Whether to replace to the HXK1 gene locus.

![](https://static.igem.org/mediawiki/2021/2/22/T--Xiamen_City--img_engineering_1.jpg)
Figure 1. Design of integrating endo-pgaA gene in wine yeast using CRISPR-Cas9 technology. P is the promoter (promotor); T is the terminator (terminator); CEP is the Cas effector protein; HR is the homology arm; endo-pgaA is the pectinase gene.

# Build

Plasmids pHCas9-Nourse and pYES2-gRNA-hyg-MCS were successfully constructed (Figure 2). The
integration of endo-pgaA gene in wine yeast genome was verified by DNA sequencing.

![](https://static.igem.org/mediawiki/2021/f/fe/T--Xiamen_City--img_engineering_2.jpg)
Figure 2. Schematic maps of plasmids pHCas9-Nourse and pYES2-gRNA-hyg-MCS.

# Test

Yeast transformants were picked pectinase production. The pectinase activity was determined using DNS
method (inducer is β-galactose) (Table 1). The specific activity was calculated from a reduce sugar
standard curve.

Table 1. Pectinase activity in yeast transformants.
![](https://static.igem.org/mediawiki/2021/c/c9/T--Xiamen_City--img_engineering_3.jpg)

# Learn

In this project, we successfully prepared genetically engineered wine yeast strain which contains
pectinase in its genome. The pectinase produced from yeast can well degrade pectin into small
sugars.

## Contact Info

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# Team:Xiamen City/Experiments

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# Experiments

# 1. Prepare competent yeast cells

1.1 Prepare yeast seed solution. (Saccharomyces cerevisiae)

* Add 1% Angel yeast (AQ-pgaA）into a 4mL test tube with liquid YPD medium.
* Mix the solution.
* Place the yeast seed solution into a shaker to incubate.

1.2 Dilute the yeast solution by a factor of 10, and measure the OD (optical density at the
wavelength of 600nm) using a spectrophotometer in order to determine the titer in cell culture.

* Pipette 100μL of yeast seed solution into 900μL sterile water

1.3 Repeatedly centrifuge the cells, remove the supernatant with a micropipette, and pipette sterile
water to re-suspend the cell pellet.

1.4 Pipette sterilized cryoprotectant into each test tube of cell pellet in the ratio of 1 to 1.

* Content of yeast cryoprotectant(10mL) :

  1) Glycerin 500μL (5%)

  2) DMSO 1000μL (10%)

  3) Sterile water 8500μL

1.5 Aliquot the solution into 10 tubes, preserve 8 of them into -20°C fridge, and use the other 2 to
transfer plasmids with Cas9 gene into competent yeast cells

# 2. Proliferate plasmids with Cas9 gene and plasmid with sgRNA gene

2.1 Add Cas9 plasmid into Escherichia coli

2.2 Add gRNA plasmid into Escherichia coli

2.3 Place the 2 tubes of solution into the water bath at 42°C for 20 minutes

2.4 Inoculate Escherichia coli with Cas9 plasmids and gRNA plasmids into LB medium with AMP
antibiotics.

* Take 100μL solution from each tube and centrifuge the solution. After that, take out 70μL
  supernatant. Apply the remaining 30μL E.coli solution to LB medium evenly.
* Place the 2 LB medium into the thermostatic incubator for 8 hours at 37℃

2.5 Prepare 2 tubes of bacterial solution

* Mix 4mL liquid LB medium with 4mL AMP antibiotics solution for each tube.
* Transfer a single E.coli bacterium with Cas9 plasmids into 1 tube, and gRNA plasmids into the
  other.

2.6 Place the 2 tubes into a shaker and incubate at 37℃

2.7 Extract Cas9 plasmids DNA and gRNA plasmids DNA from the 2 tubes using the column extraction
method.

* Discard the flow-through in the column.
* Add 500μL washing buffer to the column and centrifuge at 12,000 for 1 minute.
* Repeat the washing procedure.
* Discard the flow-through in the tube and centrifuge at 12,000rpm for an additional 2 minutes
  to remove any residual wash solution.
* Add 65 °C ddH2O (distilled water) to the tube and centrifuge at 12,000rpm for 1 minute.
* Collect the DNA fluid (Cas9 plasmid DNA, gRNA plasmid DNA) in the tube and discard the
  EZ-10.

# 3.Transform plasmids with Cas9 gene into competent yeast cells

3.1 Pipette the following reagents into the 2 tubes of cell pellet in the listed order

* Cas9 plasmid 2.5μL
* Sterile water 31.5μL
* PEG3350 240μL (sticky liquid that protects DNA from LiAC)
* ssDNA 50μL (single strand DNA, extracted from tuna, which brings Cas9 plasmid into yeast
  cells. ssDNA should be heated at 100℃ for 10min and be placed into ice immediately before added
  into the solution.)
* LiAC 36μL (catalyst)

3.2 Incubate 2 tubes of cell pellet into a water bath for 20 minutes at 42℃, allowing Cas9 plasmid to
go into the yeast cells.

3.3 Centrifuge the 2 tubes of cell pellet and remove the supernatant

3.4 Pipette 500μL of liquid YPD medium into the 2 tubes.

3.5 Prepare YPD-Nourse medium to cultivating the Cas9 yeast

* YP medium (solid) 45mL
* Glucose 5mL
* Nourse (200mg/mL) 50μL
* 20mg Nourse powder, 100μL sterile water

3.6 Pour the medium into the Petri dishes

3.7 Coat the 2 tubes of solution onto the Petri dishes evenly

3.8 Incubate the Petri dishes into the thermostatic incubator at 30°C

# 4. Prepare competent yeast cells that contain Cas9 plasmid.

4.1 Prepare yeast seed solution that contains Cas9 plasmid

* Add 4mL YPD medium liquid and 2μL Nourse antibiotic. Pick up a single bacterium which has
  grown for two days in the 30°C incubator and add it into the solution.
* Place the yeast solution into the shaker and incubate for 2-3 days.

4.2 Dilute the yeast solution by the factor of 10, and test the OD (optical density at the wavelength
of 600nm) using a spectrophotometer.

4.3 Centrifuge the yeast solution at 4500rpm for 4 minutes, and remove the supernatant.

4.4 Add 45mL of sterile water to the yeast solution, centrifuge it at 4500rpm for 4 minutes, and
remove the supernatant. (Repeat this step twice)

4.5 Add 400μL of cryoprotectant to the yeast solution.

4.6 Aliquot the yeast solution to ten 1.5ml test tubes.

4.7 Preserve the test tubes in a -20°C refrigerator.

# 5. Transform plasmids with gRNA gene and homologous recombination template into competent yeast cells that contain Cas9 plasmid.

5.1 Prepare 3 samples (upstream and downstream homology templates + PgaA gene)

PgaA sample

-Add the following reagents in descending order of volume (except sterile water)

a. 16μL sterile water

b. Fx Buffer 25μL

c. PgaA foward primer and PgaA reverse primer 10μL each

d. Diluted ten times dNTP solution 5μL

e. KOD Fx 1μL

f. PgaA template 2μL

Upstream homology template

-Add the following reagents in descending order of volume (except sterile water)

a. 16μL sterile water

b. Fx Buffer 25μL

c. upstream foward primer and upstream reverse primer 10μL each

d. Diluted ten times dNTP solution 5μL

e. KOD Fx 1μL

g. upstream homology template 1μL

Downstream homology template

-Add the following reagents in descending order of volume (except sterile water)

a. 16μL sterile water

b. Fx Buffer 25μL

c. downstream forward primer and downstream reverse primer 10μL each

d. Diluted ten times dNTP solution 5μL

e. KOD Fx 1μL

f. downstream homology template μL

5.2 Place the 3 samples in PCR apparatus

![](https://static.igem.org/mediawiki/2021/0/02/T--Xiamen_City--img_experiments_1.jpg)

5.3 After PCR, add the following reagents into the 3 samples

-For electrophoresis

Obtain 50μL of three samples relatively, and add the following reagents to each sample:

a. 60% loading buffer 10μL

b. 100% Gelred 6μL

5.4 Prepare agarose gel (2% concentratation), and place it into the horizontal agarose nucleic
acid electrophoresis tank

-Agarose 2g

-TAE buffer 200mL

5.5 Pipette 10μL of each solution into each well on the agarose gel.

5.6 After 30 minutes, cut the part of the agarose with fluorescent labelling.

5.7 Extract the DNA from the agarose gel of pgaA and upstream and downstream arm templates using the
column extraction method. (slightly different from the plasmid DNA extraction method)

-Measure the weight of the cut agarose gel.

-Add in buffer 2, which is 3 times the weight of the cut agarose gel.

-Incubate the mixture in the water bath at 50°C for 5-10 minutes.

-Move the liquified agarose gel to an absorption column, centrifuge and remove the
supernatant.

-Meanwhile, mix absolute alcohol with wash buffer, and add 500μL of the mixture into the same
absorption column. Centrifuge the absorption column for 30 seconds, and remove the supernatant.
(Repeat 2 times)

-Centrifuge the absorption column for 1 minute.

-Place the absorption column in a 1.5ml collection tube.

-Add 20μL sterile water at 65°C.

-Centrifuge the solution, and preserve the collection tube, which contains the DNA of the PCR
products.

-Repeat the above step 3 times.

5.8 Use overlap PCR to integrate the 3 pieces of DNA together into a homologous recombination
template.

-Mix the following reagents together.

![](https://static.igem.org/mediawiki/2021/c/cf/T--Xiamen_City--img_experiments_2.jpg)

-PCR setting:

![](https://static.igem.org/mediawiki/2021/9/96/T--Xiamen_City--img_experiments_3.jpg)

-During the last 5 minutes of the 68 degree Celsius step, add 2μL of primers HR-R and primer HR-L
into the tube.

-Then, continue PCR with this setting:

![](https://static.igem.org/mediawiki/2021/3/3c/T--Xiamen_City--img_experiments_4.jpg)

-Preserve the integrated homologous recombination template.

5.9 Insertion of the gRNA plasmid DNA and the homologous recombination template into the yeast
competent cells that contain the Cas9 plasmid.

-Mix the following reagents in the listed order;

-Sterile water 24.5μL

-Homologous recombination template 4.5μL

-gRNA plasmid DNA 5μL

-PEG3350 240μL (sticky liquid that protects DNA from LiAc)

-LiAc 36μL (Catalyst)

-ssDNA 50μL (single strand DNA, extracted from tuna, which brings the Cas9 plasmid into the yeast
cells. ssDNA should be heated at 100℃ for 10min and be placed into ice immediately before added
into the solution.)

-Incubate the mixture in the water bath at 42°C for 20 minutes.

5.10 Centrifuge the solution and remove the supernatant.

5.11 Add 1000μL liquid YPD medium to the solution, and place it in the shaker to incubate at 30°C for
1 hour to revive the yeast.

5.12 Centrifuge the solution and remove 800μL of the supernatant.

5.13 Coat the remaining concentrated solution onto 3 double-antibiotics mediums
(Nourse+Hygromycin)

-Prepare 3 double-antibiotics medium:

-50mL liquid YPD medium

-50μL of Nourse mother liquor (200mg/mL)

-100μL of Hygromycin B mother liquor (400mg/mL)

5.14 Wait 2-3 days for the yeast to grow.

5.14 Prepare 4 tubes of yeast solution (2 tubes for one of the medium because the yeast is growing
well) that contains Cas9 plasmid, gRNA plasmid, and the homologous recombination template.

-Liquid YPD medium 4mL

-Nourse mother liquor 2μL

-Hygromycin B mother liquor 4μL

-A single yeast from one of the 3 mediums.

5.15 Place the 4 tubes of yeast solution into the shaker to incubate for 2-3 days.

# 6. Enzyme Activity Test

6.1 Test the OD of the 4 tubes of yeast solution

-OD results: (Dilute the 4 tubes of yeast solution by pipetting 100 μL of yeast solution into
900 μL of sterile water beforehand)

-Solution 1: 0.42 x 10 = 4.2

-Solution 2: 1.10 x 10 = 11

-Solution 3: 1.14 x 10 = 11.4

-Solution 4: 1.41 x 10 = 14.1

6.2 Preserve the 4 tubes of bacterial solution by adding glycerol.

-Add 900 μL yeast solution with 900μL glycerol solution (40%)

6.3 Make 5 double-layer pectin medium (3 for the examination of congo Congo red dye, 2 for the
examination for bromophenol blue dye)

6.3.1 Compose Prepare 5 double-layer pectin medium.

-Pour in the first layer of the pectin medium, and wait for the liquid to solidify.

-Inversely stab 7 pipette tips (for 2.5μL pipette) into half the depth of the solidifieds pectin
layer.

-Pour in the second layer of the pectin medium, and wait for the liquid to solidify.

-Slowly remove the 7 pipette tips without damaging the pectin layers.

(-repeat the above steps for 3 times to compose prepare 3 double-layer pectin medium for the
examination with congo Congo red dye, which is added at the end)

(-add 0.5g bromophenol blue dye into 100mL liquid pectin medium, and repeat the above steps for 2
times to prepare 2 double-layer pectin medium for the examination with bromophenol blue dye)

(use the diluted yeast solution 1, 2, 3 for the examination of Congo red dye, only use the
diluted yeast solution 3 for the examination for bromophenol blue dye.)

![](https://static.igem.org/mediawiki/2021/6/64/T--Xiamen_City--img_experiments_5.jpg)

(-repeat the above steps for 3 times to prepare 3 double-layer pectin medium for the examination
with Congo red dye, which is added at the end)

6.4 Inoculate the yeast solution at different concentrations into each hole.

(We only used the first 3 tubes of yeast solution, because the fourth one is not enough for
inoculation)

6.4.1 Prepare a control group with Angel yeast, and dilute each of the 3 tubes of yeast solution into
6 different concentrations.

6.5 Pipette 15μL of the 8 prepared liquid into 3 double-layer pectin mediums.

6.6 Pipette 60μL of the 8 prepared liquid into 2 double-layer pectin mediums.

# 7. Induction Experiment

7.1 Inoculate AQ-pgaA glycerol bacteria into 10ml YPD medium, culture at 30℃, 220r/ml shaking for
36h, and detect the cell concentration OD600=0.6295;

7.2 Take 5ml of bacterial solution in a 50ml centrifuge tube, centrifuge at 1500g at 4℃ for 5min, and
collect the bacterial pellet;

7.3 Take 1ml of YPD induction medium containing 2% galactose to fully suspend the bacteria, and
supplement the induction medium to 50ml, and culture all the bacteria in a 250ml Erlenmeyer flask
(1:50 inoculation), 30℃, 220r/ml Shock training

# 8. DNS Test

8.1 Detection principle: pectinase hydrolyzes pectin β-galacturonic acid, and reacts with
dinitrosalicylic acid (DNS) to form a purple-red compound. The color intensity of this compound is
proportional to the concentration of galacturonic acid. The absorbance is measured by a
spectrophotometer, and the pectinase activity in the sample is calculated by comparing with the
standard curve.

8.2 Pectinase extraction: 48h sample is not broken, the supernatant is not centrifuged, and the
supernatant is centrifuged after crushing;

8.3 Dilution of standard pectinase: Take an appropriate amount of standard pectinase (1mg/ml) and
dilute:

8.4 Adding samples: add them in the order of the solutions in the table below to avoid bubbles and
mix carefully;

8.5 Determination of DNS: Take 1ml pectin standard solution and add it to a 15ml centrifuge tube, add
2ml DNS reagent, boil water bath for 5min, cool tap water to room temperature, take 1ml into a
cuvette, and measure OD value at 540nm. Take the absorbance value as the ordinate and each standard
concentration (mg/ml) as the abscissa to draw the pectin standard curve.

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# Results

# 1. Construction of CRISPR expression plasmids

The *PgaA* is an enzyme that hydrolyzes the α-1,4 glycosidic bonds between galacturonic acid
residues present in polygalacturonan in plant cell walls and therefore facilitate plant cell wall
breakdown. In the production of fruit wine, pectinase has been used to destroy the pectin in the
cell wall in order to improve the juice yield and increase the dissolution of aromatic substances
such as pigments or terpenes.

![](https://static.igem.org/mediawiki/2021/3/3e/T--Xiamen_City--img_results_1.jpg)

**Fig.1 Construction CRISPR expression plasmids.** (A) Schematic
representation of CRISPR expression vectors; (B) Agarose gel electrophoresis of pHCas9-Nours
(lanes 1 and 2) and pYES2–gRNA-hyg-MCS (lanes 3 and 4) plasmids.

We sought to integrate *PgaA* gene into *S. cerevisiae* genome through CRISPR technology in
order to obtain the yeast strains that not only produce alcohol but can also decompose pectin. To
this end, we designed two plasmids expressing Cas9 and gRNA (Fig. 1A), as well as the repair
template. The agarose gel electrophoresis results indicated that the plasmids of pHCas9-Nours and
pYES2–gRNA-hyg-MCS were extracted from DH5 with high quality and could be used for the following
transformation experiments.

# 2. Construction of the repair template

The repair template DNA containing PgaA gene (Fig. 2A) was generated by the overlap-PCR method.
Firstly, the DNA fragments of upstream and downstream homologous regions were amplified with ~20 bp
ends overlapping to the PgaA gene, producing ~500 bp PCR products (Fig. 2B). Secondly, the two
fragments were annealed to the 5’- and 3’-ends of PgaA. Finally, the annealed products were further
amplified using end primers of HR-L and HR-R, resulting in a DNA fragment of 1.5 kbp, which was
verified as correct by agarose gel electrophoresis and DNA sequencing (Figs. 2C and 2D).

![](https://static.igem.org/mediawiki/2021/c/cf/T--Xiamen_City--img_results_2.jpg)

**Fig. 2 Construction of repair template.** (A) Schematic
representation of repair template; (B) Agarose gel electrophoresis of PCR products; (C) DNA
sequencing result analysis.

# 3. Yeast strain transformation and positive transformants verification

The constructed CRISPR plasmids and repair template DNA were chemically transformed into the *S.
cerevisiae* strains. The positive transformants were selected against YPD medium supplemented
with Nours and hygromycin. The resulting colonies were picked up and cultured. To investigate
whether the PgaA gene was integrated into yeast genome, we performed colony PCR using the upstream
and downstream primers complementary to HR-L and HR-R genes, respectively. As shown in Fig. 3A, the
target PCR products at~1500 bp were then extracted and purified for sequencing. The sequencing
results finally confirmed that the PgaA gene was successfully integrated into *S. cerevisiae*
genome (Fig. 3B).

![](https://static.igem.org/mediawiki/2021/1/19/T--Xiamen_City--img_results_3.jpg)

**Fig. 3 Verification of PgaA containing transformants.** (A)
Agarose gel electrophoresis of PCR products; (B) DNA sequencing result analysis.

To obtain an optimal culture time, we monitored the growth rate of recombinant *S. cerevisiae*
cells from 2 to 72 h. As shown in Table.1 and Fig. 4, the OD600 of culture increased from 2 h and
reached a plateau at 48 h, indicating that the optimal culture time was 48 h.

Table. 1 Growth rate of PgaA expressing cells
![](https://static.igem.org/mediawiki/2021/2/2e/T--Xiamen_City--img_results_4.jpg)

![](https://static.igem.org/mediawiki/2021/8/86/T--Xiamen_City--img_results_5.jpg)
Fig 4. Growth rate of PgaA expressing cells

# 4. Pectinase activity assay

The pectinase activities of PgaA were determined using the dinitrosalicylic acid (DNS) colorimetric
method. Briefly, in the presence of PgaA, pectin can be degraded into galacturonic acids, which
reacts with DNS to form a compound with a maximum absorption at 540 nm. Thus, the activity of PgaA
can be calculated by measuring the absorbance of the reactants with a spectrophotometer. For
accurate quantification, a standard curve was generated using a series of concentrations of
pectinase standards. As shown in Table. 2 and Fig. 5, the concentration of enzyme correlates well
with the absorbance detected at 540 nm, applying to the Lambert-Beer law.

Table. 2 Measurement of standard pectinase activities at different concentrations
![](https://static.igem.org/mediawiki/2021/8/87/T--Xiamen_City--img_results_6.jpg)

![](https://static.igem.org/mediawiki/2021/8/81/T--Xiamen_City--img_results_7.jpg)
Fig. 5 Standard curve of pectinase.

With this standard curve, we next determined the concentration of PgaA from recombinant ***S.
cerevisiae* strains**. Samples from the culture media, total cell lysates and the soluble
portion of cell lysates
were collected and subjected to DNS colorimetric assay. As shown in Table. 3, the concentration of
PgaA in the culture media of samples -1 and -2 were determined at about 0.034 mg/ml and 0.028 mg/ml,
respectively, which were relatively higher than that of cell lysates (0.009 mg/ml and 0.007 mg/ml),
suggesting that most of the PgaA proteins were secreted into the culture media. In addition, in the
cell lysates of sample 1, we detected ~76% of PgaA in the soluble supernatants, implying that most
of the PgaA in cells are soluble. Unexpectedly, the concentration of PgaA in the soluble
supernatants of sample 2 was higher than that of total cell lysates, this could be due to
experimental error. Since we measured the Culture media 2 first which possesses higher concentration
of PgaA then the Soluble portion of cell lysate 2, the ultrasonic probe might haven’t been fully
cleaned which would result the measurement error about the concentration of PgaA in the Soluble
portion of cell lysate 2.

Table. 3 Measurement of PgaA concentration and unit of activity in various samples
![](https://static.igem.org/mediawiki/2021/d/db/T--Xiamen_City--img_results_8.jpg)

#

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