iGEM 2021 — Team Beijing_United

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# Team:Beijing United/Notebook

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# Notebook

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# Team:Beijing United/Engineering

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# Engineering

# Background

China is one of the oldest countries in the world to cultivate wheat, and wheat is also one of
China's three major food crops (rice, wheat, and corn). There are usually two types of industrially
produced wheat starch, A-type and B-type. A starch is a refined starch and B starch is a by-product
of the production of A starch. The non-starch polysaccharides (pentosans, β-glucans) in B starch
cannot be decomposed by the endogenous digestive enzymes of monogastric animals. It enters the large
intestine directly and is decomposed and fermented by microorganisms in the large intestine, which
usually causes nutritional disorders in monogastric animals, especially poultry.

Alcohol (ethanol) is an important chemical in the fields of food, medicine, and biofuels. The
traditional fermentation method uses corn or tapioca starch as raw material to produce alcohol.

Wheat B starch is a by-product of deep processing of wheat starch. It is often used directly as feed,
and the added value of the industry is not high. If wheat B starch is used as a raw material to
produce alcohol, some of the disadvantages of wheat starch alcohol can be avoided and the
utilization value of wheat B starch can be improved. After wheat B starch is pretreated by liquid
saccharification to produce sugar, it can be fermented with conventional *Saccharomyces
cerevisiae* to
produce alcohol. However, the pentosan component of wheat B starch is not used in this process.
Therefore, the ideal approach is to develop *Saccharomyces cerevisiae* strains which can secret
pentosanase.

# Design

We selected codon-optimized xylanase gene AnXlnB, β-xylosidase gene AnXlnD (from *Aspergillus
niger*)
and acetyl xylan esterase CcXynA (from *Clostridium cellulophilum*). To express these gens in
*Saccharomyces cerevisiae* strain, generating a genetically modified *S. cerevisiae* with
the ability to
decompose and utilize pentosan. The ability of the strain to ferment pentosan to produce alcohol was
tested in a specific medium. The overall design is shown in Figure 1.

![](https://static.igem.org/mediawiki/2021/9/9e/T--Beijing_United--img_engineering_1.jpg)
Figure 1. Genetically modified *S. cerevisiae* with the ability to decompose and utilize pentosan, and to ferment pentosan to produce alcohol.

# Build

AnXlnB, AnXlnD, and CcXynA were inserted into yeast expression vector (Figure 2).

![](https://static.igem.org/mediawiki/2021/8/84/T--Beijing_United--img_engineering_2.jpg)
Figure 2. Plasmid maps in the project.

# Test

The xylan utilization *S. cerevisiae* strain construction and fermentation test (Figure 3).

![](https://static.igem.org/mediawiki/2021/8/82/T--Beijing_United--img_engineering_3.jpg)
Figure 3. Fermentation performance of the plasmids transformed *S. cerevisiae* strains in the simulated wheat B starch medium. A: OD value. B: Sugar concentration.

The plasmids pXylan-B and pXylan-BD were transformed into the S. cerevisiae strain, respectively. The
resulting positive transformants were undergo the fermentation test. In the simulated wheat B starch
medium (YPD20Xylan20), all the strains showed almost the same growth performance during the first 8
h, this is due to the strains preferentially utilized the glucose present in the media. This was
verified again in Figure 7B, all the strains showed the comparable sugar utilization capacity, the
xylan utilization ability may be covered by the glucose. Therefore, to verify the strains’ xylan
utilization capacity, a xylan as the sole carbon source medium was essential in further study.

The sugar consumption data showed that starting from 2 hours, the WXA/pXylan-B and WXA/pXylan-BD
strain was slightly higher than the WXA control, which could be interpreted as decomposing xylan and
producing reducing sugars.

# Learn

The engineered bacteria we constructed can decompose the xylan successfully.

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# Team:Beijing United/Experiments

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# Experiments

# Plasmid construction:

The first stage: construct plasmid pOdd-1, pOdd-2, pOdd-3 at the same time

The second stage: pOdd-1 and XynA connection to obtain pXlnA, pOdd-2 and XynB connection to obtain
pXlnB, pOdd-3 and XynD connection to obtain pXlnD

The third stage: construct pXylan-B, pXylanBD, pXylaN (\*N=BDA)

The fourth stage: transformation of yeast. The obtained pXylan-B, pXylanBD, and pXylaN were extracted
from E. coli and transformed into yeast for the next functional test.

Function test: Fermentation performance test-test sugar and alcohol production capacity

# PCR experiment:

PCR experiment can be divided into three steps: denaturation → annealing → extension. Denaturation:
heating breaks the hydrogen bond between double strands of template DNA at a high temperature of 95
℃ to form two single strands. Annealing: reduce the solution temperature by 60 ℃, and make the
template DNA and primer complement each other according to the principle of base pairing. Extension:
when the reaction temperature of the solution rises to 72 ℃, the heat-resistant DNA polymerase takes
the single strand DNA as the template and replicates the complementary DNA in the direction of 5 '-
3' by using four kinds of deoxynucleoside triphosphate (dNTP) in the reaction mixture under the
guidance of primers. We also did pre denaturation and complete extension to completely denature and
untie the primers, so as to ensure that the primers are single stranded DNA, so as to ensure the
effect. The final extension is to fully extend the primers and completely anneal the single stranded
products to form a double stranded structure to ensure the effect.

# Gel electrophoresis experiment:

TAE buffer was added and agarose gel was added to the template, waiting to be condensed into solid,
then put into TAE buffer and added DNA reagent of mixed nucleic acid dye into the gel
electrophoresis apparatus to wait for the result. The results can be seen visually under UV
lamp.

# LB medium:

The medium is used to pre culture the strains to multiply the strains and meet the use requirements.
It can also be used to culture genetically engineered receptor bacteria. We cultured E. coli.

# Plasmid transformation by heat shock method:

Plasmid transformation refers to the process of introducing our plasmid into bacteria. The connected
products were put into competent cells. It is convenient for subsequent molecular operation. To
achieve the proliferation of recombinant clones.

# Hygromycin plate screening:

The plate contains hygromycin, and our plasmid has the function of anti hygromycin. Only the yeast
transferred into our plasmid can grow on the plate. At the same time, many methods can be used to
screen and identify the target clones.

# pXlnB plasmid construction:

pXlnB is GAP promoter+AnXlnB orf+CYC1 terminator transcription unit plasmid.

1. GAP promoter part acquisition: Using Saccharomyces cerevisiae DNA (or colony) as a template
   and primer pair GAPp-F1/GAPp-R1 to amplify the GAP promoter part, with a size of 695bp.
2. AnXlnB orf part acquisition: Using the synthesized AnXlnB orf sequence as a template and the
   primer pair AnXlnBorf-F1/AnXlnBorf-R1 to amplify to obtain AnXlnB orf part with a size of
   706bp.
3. Obtained CYC1 terminator part: Using Saccharomyces cerevisiae DNA (or colony) as template and
   primer pair CYC1t-F1/CYC1t-R1 to amplify to obtain CYC1 terminator part. The size is 276bp.
4. pOdd-1 plasmid acquisition: Using pAR318 plasmid as template and primer pair
   pOdd-1-F1/pOdd-1-R1 to amplify to obtain 1757bp fragment. Then use this fragment as template and
   pOdd-1-F2/pOdd-1-R2 primer to amplify to obtain a 1804bp fragment. This 1804bp fragment has
   repetitive sequences at both ends. The linear fragment can be ligated into a circular plasmid
   using Gibson or a recombination kit (Novizan), and screened on a kanamycin plate.
5. The obtained pOdd-1 plasmid, GAP promoter part, AnXlnB orf part, CYC1 terminator part were
   cloned by Bsa1 restriction enzyme digestion and ligation, and kanamycin plate screening was
   performed. The final plasmid was named pXlnB.

# pXlnD plasmid construction:

1. pXlnD is a TPI1 promoter+AnXlnD orf+CYC1 terminator transcription unit plasmid.
2. TPI1 promoter part acquisition: Use Saccharomyces cerevisiae DNA (or colony) as a template
   and primer pair TPI1p-F1/TPI1p-R1 to amplify the TPI1 promoter part, which is 614bp in size.
3. AnXlnD orf part acquisition: Use the synthesized AnXlnD orf sequence as a template and the
   primer pair AnXlnDorf-F1/AnXlnDorf-R1 to amplifyto obtain AnXlnD orf part with a size of
   2443bp.Obtained CYC1 terminator part: Using Saccharomyces cerevisiae DNA (or colony) as template
   and primer pair CYC1t-F1/CYC1t-R1 to amplify to obtain CYC1 terminator part. The size is
   276bp.
4. pOdd-2 is the TPI1 promoter+AnXlnD orf+CYC1 terminator transcription unit receiving plasmid:
   the pAR318 plasmid is used as the template, and the primer pair pOdd-2-F1/pOdd-2-R1 is amplified
   to obtain a 1757bp fragment. Use this fragment as a template, pOdd -2-F2/pOdd-2-R2 primer to
   amplify1804bp fragment. This 1804bp fragment has repetitive sequences at both ends. The linear
   fragment can be ligated into a circular plasmid using Gibson or a recombination kit (Novizan),
   and screened on a kanamycin plate.
5. The obtained pOdd-2 plasmid, TPI1 promoter part, AnXlnD orf part, CYC1 terminator part were
   cloned by Bsa1 restriction enzyme digestion and ligation, and kanamycin plate screening was
   performed. The final plasmid was named pXlnD.

# pXynA plasmid construction:

1. pXynA is FBA1 promoter+AnXlnD orf+CYC1 terminator transcription unit plasmid.
2. Obtained FBA1 promoter part: Use Saccharomyces cerevisiae DNA (or colony) as template and
   primer pair FBA1p-F1/FBA1p-R1 to amplify to obtain FBA1 promoter part, with a size of 848bp.
3. Obtained CcXynA orf part: Use the synthesized CcXynA orf sequence as a template and the
   primer pair CcXynA orf-F1/ CcXynA orf-R1 to amplify to obtain the CcXynA orf part with a size of
   1591bp.
4. Obtained CYC1 terminator part: Use Saccharomyces cerevisiae DNA (or colony) as template and
   primer pair CYC1t-F1/CYC1t-R1 to amplify to obtain CYC1 terminator part, the size is 276bp.
5. pOdd-3 plasmid acquisition: Use pAR318 plasmid as template and primer pair
   pOdd-3-F1/pOdd-3-R1 to amplify to obtain 1757bp fragment. Then use this fragment as template and
   pOdd-3-F2/pOdd-3-R2 primer to amplify to obtain a 1804bp fragment. This 1804bp fragment has
   repetitive sequences at both ends. The linear fragment can be ligated into a circular plasmid
   using Gibson or a recombination kit (Novizan), and screened on a kanamycin plate.

The obtained pOdd-3 plasmid, FBA1 promoter part, CcXynA orf part, CYC1 terminator part were subjected
to one-step cloning with Bsa1 restriction enzyme digestion, and kanamycin plate screening, and the
final plasmid was named pXynA.

![](https://static.igem.org/mediawiki/2021/9/99/T--Beijing_United--img_experiment_1.jpg)

# Transformation

The ligation product is transformed into DH5a competent cells and screened on LB/kana plate

# Colony PCR System

Identification primer cx-podd-kana-F/ CYC1t-R1, 2\*mix, 55 degrees annealing, 72 degrees extension for
4 minutes

Send to company for sequencing

# pXylan-B plasmid construction:

Use a double-site gRNA expression plasmid as a template (such as X-3 XI-2 gRNA-hyg plasmid) and the
primer pair Backbone-XlnB-F1/Backbone-R1 to amplify the 5479bp Backbone-XlnB fragment, which is
recovered from the gel and digested with Sap1 for use.

The pXlnB plasmid was digested with Sap1 to obtain a 1624+1727bp fragment, and the 1624bp fragment
was recovered. If the two bands are indistinguishable, design primers based on the plasmid map to
obtain the GAP promoter+AnXlnB orf+CYC1 terminator transcription unit in pXlnB plasmid, and then use
Sap1 restriction enzyme digestion.

The above two fragments, namely the Backbone-XlnB fragment after Sap1 digestion, and the 1624bp
fragment, were ligated and cloned by restriction enzyme digestion, and screened on ampicillin
plates. Finally, the plasmid pXylan-B is obtained.

# pXylan-BD plasmid construction:

Use a double-site gRNA expression plasmid as a template (such as X-3 XI-2 gRNA-hyg plasmid) and the
primer pair Backbone-XlnBD-F1/Backbone-R1 to amplify the 5479bp Backbone-XlnB fragment, which is
recovered by gel and digested with Sap1 for use.

The pXlnB plasmid was digested with Sap1 to obtain a 1624+1727bp fragment, and the 1624bp fragment
was recovered. If the two bands are indistinguishable, design primers based on the plasmid map to
obtain the GAP promoter+AnXlnB orf+CYC1 terminator transcription unit in pXlnB plasmid, and then use
Sap1 restriction enzyme digestion.

The pXlnD plasmid was digested with Sap1 to obtain a 3280+1727bp fragment, and the 3280bp band was
recovered for use.

The above three fragments, namely the Backbone-XlnBD fragment after Sap1 digestion, the 1624bp
fragment, and the 3280bp fragment, were cloned by restriction digestion and ligated in one step, and
screened on ampicillin plates. Finally, the plasmid pXylan-BD was obtained.

# pXylan plasmid construction:

Use a double-site gRNA expression plasmid as a template (such as X-3 XI-2 gRNA-hyg plasmid). The
primer pair Backbone-F1/Backbone-R1 was used to amplify the 5479bp Backbone fragment, which was
recovered from the gel, and digested with Sap1 for use.

The pXlnB plasmid was digested with Sap1 to obtain a 1624+1727bp fragment, and the 1624bp fragment
was recovered. If the two bands are indistinguishable, design primers based on the plasmid map to
obtain the GAP promoter+AnXlnB orf+CYC1 terminator transcription unit in pXlnB plasmid, and then use
Sap1 restriction enzyme digestion.

The pXlnD plasmid was digested with Sap1 to obtain a 3280+1727bp fragment, and the 3280bp band was
recovered for use.

The pXynA plasmid was digested with Sap1 to obtain a 2662+1727bp fragment, and the 2662bp band was
recovered for use.

The above 4 fragments, namely the Backbone fragment after Sap1 digestion, the 1624bp fragment, the
3280bp fragment, and the 2662bp fragment, were cloned by restriction enzyme digestion and ligated in
one step, and screened on an ampicillin plate. Finally, the plasmid pXylan was obtained.

# DNS method for the determination of reducing sugars

Steps: Draw 1 mL of sample solution. Put it in a 25 mL colorimetric tube. Add 2 mL of DNS reaction
solution, and develop color in boiling water for 2 minutes. Rinse with running water, after cooling
quickly, add 9 mL of distilled water to dilute, and shake well. Adjust to zero with distilled water
blank, measure the absorbance at 540 nm, and calculate the amount of reducing sugar by referring to
the standard curve.

The preparation of the standard curve: the standard glucose is first dried to a constant weight,
accurately weighed 1 g, dissolved in a small amount of distilled water, and dilute to 500 mL, so
that the reducing sugar concentration is 2 mg/mL. Pipette 0.2, 0.4, 0.5, 0.6, 0.8 mL of standard
solution into 25 mL colorimetric tube. Make up to 1 mL with distilled water. Add 2 mL of DNS
reaction solution, and develop color with boiling water for 2 minutes. Rinse with running water, and
quickly cool down. Dilute with 9 mL of distilled water and shake well. Set the blank with distilled
water to zero, and measure the absorbance at 540 nm. Draw the standard curve with the absorbance
value OD540 at 540 nm as the abscissa and the reducing sugar content C (g/L) as the ordinate.

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# Team:Beijing United/Results

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# Results

## 1. *Aspergillus niger* derived xylanase expression plasmid construction

![](https://static.igem.org/mediawiki/2021/d/dc/T--Beijing_United--img_proof_of_concept_1.jpg)
Figure 1 pXlnB and pXylan-B plasmids map

To utilize the xylan component contained in the wheat B starch, we cloned the xylanase expression
gene from *Aspergillus niger*. The xylanase expression cassette contained pXlnB plasmid was
constructed firstly to prepare the final plasmid pXylan-B (Figure 1).

![](https://static.igem.org/mediawiki/2021/b/b1/T--Beijing_United--img_proof_of_concept_2.jpg)
Figure 2 Plasmids construction used fragments PCR amplification. (A) Lane 1: GAP promoter, 695 bp. Lane 2: *AnXlnB* CDS, 706 bp. Lane 3: CYC1 terminator, 276bp. Lane 4: pXlnB plasmid backbone fragment, 1757 bp. Lane 5: TPI1 promoter, 614 bp. Lane 6: *AnXlnD* CDS, 2443 bp. Lane 7: pXlnD plasmid backbone fragment, 1804 bp. (B) Lane 1: pXlnB plasmid backbone fragment, 1804 bp. Lane 2: pXlnD plasmid backbone fragment, 1804 bp. (C) Lane 1: pXylan-B plasmid backbone, 5479 bp. Lane 2: pXylan-BD plasmid backbone, 5479 bp.

For the pXlnB plasmid construction, the promoter GAP, codon-optimized *AnXlnB* CDS, and CYC1
terminator
PCR bands were shown in the Figure 2A, lane 1, lane2, and lane 3, respectively. The
*AnXlnB*
expression cassette was obtained through the overlap PCR. The backbone fragment (kanR with ori)
was
amplified using two round PCR, the first round and the final fragment band were shown in Figure
2A
lane 4 and Figure 2B lane 1, respectively. The backbone was cut with Bsa1 restriction enzyme and
ligated with the *AnXlnB* expression cassette to make the plasmid pXlnB.

For the construction of the final plasmid pXylan-B, the pXlnB was cut with *Sap1*
restriction
enzyme,
and the backbone part (Figure 2C) was also cut with the same enzyme, these two parts were
ligated to
make the final plasmid pXylan-B.

## 2. *Aspergillus niger* derived xylanase and *β-xylosidase* expression plasmid construction

![](https://static.igem.org/mediawiki/2021/4/45/T--Beijing_United--img_proof_of_concept_3.jpg)
Figure 3 pXlnD and pXylan-BD plasmids map

For the effective utilization of the xylan component present in the wheat B starch, the *Aspergillus
niger* derived *β-xylosidase* (Figure 3A) was also cloned together with the xylanase
expression gene.
To make the final plasmid pXylan-BD (Figure 3B), the pXlnD plasmid was constructed firstly. The
promoter TPI1, codon-optimized *AnXlnD* CDS, and CYC1 terminator PCR bands were shown in
the
Figure
2A, lane 5, lane6, and lane 3, respectively. The *AnXlnD* expression cassette was obtained
through the
overlap PCR. The backbone fragment (kanR with ori) was amplified using two round PCR, the first
round and the final fragment band were shown in Figure 2A lane 7 and Figure 2B lane 2,
respectively.
The backbone was cut with Bsa1 restriction enzyme and ligated with the *AnXlnD* expression
cassette to
make the plasmid pXlnD.

For the construction of the final plasmid pXylan-BD, the pXlnB and pXlnD were both cut with Sap1
restriction enzyme, and the backbone part (Figure 2C) was also cut with the same enzyme, these
three
parts were ligated to make the final plasmid pXylan-BD.

![](https://static.igem.org/mediawiki/2021/0/0b/T--Beijing_United--img_proof_of_concept_4.jpg)
Figure 4 Positive colonies verification through the colony PCR. Lane 11 to 20, pXlnB plasmid colony PCR verification. Lane 21 to 30, pXlnD plasmid colony PCR verification.

Figure 4 demonstrated the positive colonies verification of the plasmids pXlnB and pXlnD. The
number
of 12 to 16, 18 to 20 were the positive colonies of the plasmid pXlnB, the number of 21, 23, 24,
27
to 30 were the positive colonies of the plasmid pXlnD. Number 12 of pXlnB and number 23 of pXlnD
were sent for the sequencing.

![](https://static.igem.org/mediawiki/2021/4/4d/T--Beijing_United--img_proof_of_concept_5.jpg)
Figure 5 Plasmids DNA sequencing. A: pXlnB plasmid. B: pXlnD plasmid.

The positive transformants verified using the colony PCR were sent for the DNA sequencing, as
shown
in Figure 5, both the plasmids pXlnB and pXlnD were constructed successfully.

![](https://static.igem.org/mediawiki/2021/c/c5/T--Beijing_United--img_proof_of_concept_6.jpg)
Figure 6 Positive colonies verification through the colony PCR (A) and DNA sequencing of the plasmid pXylan-B (B) and pXylan-BD (C).

Figure 6A demonstrated the positive colonies verification of the plasmids pXylan-B and pXylan-BD.
The
numbers 10, 12, and 14 were the positive colonies of the plasmid pXylan-B, the numberss 2, 4,
and 6
were the positive colonies of the plasmid pXylan-BD. Number 12 of pXylan-B and number 4 of
pXylan-BD
were sent for the sequencing. Figures 6B and C showed that both the pXylan-B and pXylan-BD
plasmids
were constructed successfully.

## 3. The xylan utilization Saccharomyces cerevisiae strain construction and **fermentation test**

![](https://static.igem.org/mediawiki/2021/8/88/T--Beijing_United--img_proof_of_concept_7.jpg)
Figure 7 Fermentation performance of the plasmids transformed *S. cerevisiae* strains in the simulated wheat B starch medium. A: OD value. B: Sugar concentration.

The plasmids pXylan-B and pXylan-BD were transformed into the *S. cerevisiae* strain,
respectively. The
resulting positive transformants were used in the fermentation test. In the simulated wheat B
starch
medium (YPD20Xylan20), all the strains **showed almost the same growth performance during the
first
8
h**, this is due to the strains preferentially utilized the glucose present in the media.
This
was
verified again in Figure 7B, all the strains showed the **comparable sugar utilization
capacity**,
the
xylan utilization ability may be covered by the glucose. Therefore, to verify the strains’ xylan
utilization capacity, a xylan as the sole carbon source medium was essential in further study.

The sugar consumption data showed that starting from 2 hours, the WXA/pXylan-B and WXA/pXylan-BD
strain was slightly higher than the WXA control, which could be interpreted as decomposing xylan
and producing reducing sugars. Therefore, the engineered bacteria we constructed can decompose
the xylan successfully.

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