iGEM 2024 — Team fdfzsh

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each
day for your project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5351/description.png)
![](https://static.igem.wiki/teams/5351/description-c.png)
![](https://static.igem.wiki/teams/5351/description-b.png)
![](https://static.igem.wiki/teams/5351/description-a.png)
![](https://static.igem.wiki/teams/5351/ahaystack.png)
NOTEBOOK

# NOTEBOOK

### （Wet Lab Notebook）

### Main Timeline:

（Each month is depicted with a grid system where each grid corresponds to 3 days. The shaded regions indicate the
days
designated for work.）

### January:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Establishment of the project team Starting from 11th |  |  |  | Starting from 11th |  |  |  |  |  |  |
| Development of Project Plan |  |  |  | Starting from 11th |  |  |  |  |  |  |
| Recruiting of new members |  |  |  | Starting from 11th |  |  |  |  |  |  |

### February:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Establishment of the project team |  |  |  |  |  |  |  |  |  |  |
| Development of Project Plan |  |  |  |  |  |  |  |  |  |  |
| Recruiting of new members |  |  |  |  |  |  |  |  |  |  |

### March:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Establishment of the project team |  |  |  |  |  |  |  |  |  |  |
| Development of Project Plan |  |  |  |  |  |  |  |  |  |  |

### April:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Establishment of the project team |  |  |  |  |  |  |  |  |  |  |
| Development of Project Plan |  |  |  |  |  |  |  |  |  |  |

### May:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Subject determination |  |  |  |  |  |  |  |  |  |  |
| Theoretical study |  |  |  |  |  |  |  |  |  |  |

### June:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Theoretical study |  |  |  |  |  |  |  |  |  |  |
| Subject determination |  |  |  |  |  |  |  |  |  |  |
| Experimental design |  |  |  |  |  |  |  |  |  |  |
| Safety training |  |  |  |  |  |  |  |  |  |  |
| Experimental design |  |  |  |  |  |  |  |  |  |  |

### July:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Experimental design |  |  |  |  |  |  |  |  |  |  |
| Problems discussion |  |  |  |  |  |  |  |  |  |  |
| Record experiment report |  |  |  |  |  |  |  |  |  |  |
| Building the Team Wiki |  |  |  |  |  |  |  |  |  |  |

### August:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Implement experimental plan |  |  |  |  |  |  |  |  |  |  |
| Problems discussion |  |  |  |  |  |  |  |  |  |  |
| Analysis of experimental results |  |  |  |  |  |  |  |  |  |  |
| Building the Team Wiki |  |  |  |  |  |  |  |  |  |  |

### September:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Problems discussion |  |  |  |  |  |  |  |  |  |  |
| Building the Team Wiki |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |
| Preparation of defense materials |  |  |  |  |  |  |  |  |  |  |
| Analysis of experimental results |  |  |  |  |  |  |  |  |  |  |

### October:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Preparation of defense materials |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |
| Project review |  |  |  |  |  |  |  |  |  |  |
| Building the Team Wiki |  |  |  |  |  |  |  |  |  |  |

### Record of Main Experimental Content

### Pre-experiment safety training:

### The main content is as follows:

I. Always ensure to wear the necessary personal protective equipment, including lab coats, gloves, and safety
goggles,
when working in the laboratory.

II. Be familiar with the location and proper usage of emergency equipment, such as eyewash stations, fire
extinguishers, and emergency showers.

III. Refrain from consuming food or beverages or applying cosmetics in the laboratory to avoid accidental exposure
to
hazardous substances.

IV. Adhere to correct procedures for handling and disposing of chemicals, biohazardous materials, and sharps to
reduce
potential risks.

V. Maintain cleanliness and organization in work areas to prevent accidents and the spread of contaminants.

VI. Promptly report any spills, accidents, or injuries to the laboratory supervisor.

VII. Refrain from conducting any unauthorized activities or experiments without appropriate training and
supervision.

VIII. Utilize equipment and instruments solely for their designated purposes and operate them following safety
protocols.

IX. Acquaint yourself with emergency protocols, including evacuation routes and assembly points, in the event of
emergencies.

X. Attend regular laboratory safety training sessions and strictly adhere to safety protocols and guidelines to
ensure
a secure working environment for both yourself and others.

### Part 1:

I: Amplify the target gene by PCR, verify band size through agarose gel electrophoresis, recover the target gene
from
the gel, and store it at -20°C for future use.

II: Heat shock the recombinant product into Escherichia coli DH5a, plate on antibiotic-resistant agar plates, and
incubate at 37°C.

III: Inoculate the shaken culture with X-3 and XI-2 plasmid backbones and culture overnight at 37°C.

#### Detailed records of the main experiments: I: The basic steps of PCR technology for amplifying the target gene include denaturation, annealing, and extension. Denaturation: Initially, DNA is heated to 90-95°C, breaking the hydrogen bonds of the double-stranded DNA template to form single-stranded DNA. This step prepares for the subsequent annealing and extension processes. Annealing: Subsequently, the temperature is lowered to 55-60°C, allowing the primers to bind to the single-stranded DNA template, forming localized double strands. This step utilizes the principle of base pairing to facilitate the specific binding of primers to the target region of the DNA template. Extension: Finally, the temperature is raised again to 70-75°C, where Taq polymerase (optimal activity around 72°C) utilizes dNTPs to extend from the 5′ to 3′ end of the primer, synthesizing a complementary DNA strand to the template. This step completes the synthesis of a new DNA strand, thereby achieving DNA replication. Each cycle involves denaturation, annealing, and extension, doubling the DNA content. By repeating this process multiple times (usually 25-30 cycles), specific amplification of a particular DNA fragment can be achieved. This PCR process is based on the fundamental principle of DNA replication, allowing for the extensive replication of the target DNA sequence through repetitive cycling to meet subsequent experimental or application needs. II: Preparation of medium Yeast extract peptone dextrose YPD/X medium for yeast cultivation. The concentrations of yeast extract and peptone are 10 g/L and 20 g/L, respectively, and they are separately sterilized with 400 g/L glucose or xylose solutions. After sterilization, the calculated glucose or xylose solution is added to YP to achieve a final concentration of 20 g/L. The preparation of YPD/X solid medium only requires the addition of 2% (w/v) agar during YP sterilization. Part 2: I: Verify pscm-ISU1 and pscm-nfsI colonies by colony PCR and confirm target bands through agarose gel electrophoresis. II: Perform overlap PCR amplification of TEF1-XI-ADH1 and GAP-XI-CYC1, verify band sizes through agarose gel electrophoresis, and proceed with DNA gel extraction. III: Extract X-3 and XI-2 backbone plasmids, perform double enzyme digestion with XhoI and MssI, and proceed with DNA gel extraction. IV: T4 ligation of TEF1-XI-ADH1 with X-3 and TEF1-XI-ADH1 with XI-2, incubate at 37°C for 2-3 hours. V: Heat shock the recombinant products X-3-XI and XI-2-XI into Escherichia coli DH5a, plate on Amp plates, and culture overnight at 37°C. VI: Shake-culture X-3-XI and XI-2-XI strains. Detailed records of the main experiments: Colony PCR involves several key steps, including primer design, colony picking, PCR reaction, and result analysis. Primer Design: The first step in colony PCR is primer design, which includes three strategies: insert-specific primers, vector-specific primers, and directional-specific primers. Primer design is crucial in colony PCR as it directly impacts the specificity and efficiency of the PCR reaction. Insert-specific primers are annealed to the insert-specific sequence, vector-specific primers are annealed to the vector sites flanking the insert, and directional-specific primers determine the orientation of the insert. Before screening colonies with colony PCR primers, it is essential to test them. One recommended method is to use the target fragment primers on one end and validation primers on the vector backbone on the other end to confirm the successful construction of the fusion vector. Colony Picking: Use a sterile toothpick or pipette tip to pick a single colony and spot it on a selective plate for clonal preservation (labelling). Then, place the toothpick or pipette tip with the colony into a PCR tube containing the PCR mix to release the cells into the PCR mix as a template. PCR Reaction: Put the PCR mix containing the bacterial cells into a PCR machine and amplify following standard conditions. The PCR reaction typically includes Taq buffer, dNTPs, forward and reverse primers, polymerase, etc., with specific components and concentrations adjusted based on experimental requirements and optimization conditions. Result Analysis: After PCR amplification, analyze the PCR products using agarose gel electrophoresis to visualize the target bands. If the band size matches the expected size, it indicates the presence of the target gene or plasmid in the colony, confirming a positive clone. Additionally, sterile techniques, contamination prevention, primer specificity, and efficiency considerations in primer design are crucial in colony PCR to ensure the accuracy and reliability of experimental results. Part 3: I: Verify X-3-XI and XI-2-XI colonies through colony PCR and confirm band sizes by agarose gel electrophoresis. II: Extract X-3-XI and XI-2-XI plasmids, perform double enzyme digestion on the plasmids, and recover the target bands from the gel. III: Perform T4 ligation of the vector and target gene fragments, followed by heat shock transformation into Escherichia coli. IV: Shake-culture the phcas9 and gRNA strains. V: Prepare YPD medium for bacterial culture, sterilize, and set aside. Detailed records of the main experiments: The key steps in microbial inoculation and cultivation typically include the following: Sterilization: First, all tools and containers used need to undergo sterilization to prevent contamination by external microorganisms. This includes inoculation loops, needles, culture media, and petri dishes. Inoculation: After sterilization, a small amount of the original microbial culture is transferred using an inoculation loop or needle and then inoculated onto the culture medium. There are various inoculation methods, such as streaking and spread plating, with the specific method depending on the experimental purpose and microbial characteristics. Cultivation: After inoculation, the petri dishes are placed at the appropriate temperature for cultivation. Most microorganisms are typically cultivated at 37°C for 18-24 hours, but the exact temperature and time may vary depending on the type of microorganism. Observation and Recording: During cultivation, it is important to regularly observe the growth of microorganisms, including the morphology, color, and size of colonies, and record these observations. This helps in subsequent identification and applications. Throughout the entire process, the aseptic technique is crucial, as even minor contamination can lead to experimental failure. Therefore, strict adherence to aseptic techniques from sterilization to vaccination is essential. Part 4: I: Verify X-3-2XI and XI-2-2XI colonies through colony PCR and confirm band sizes by agarose gel electrophoresis. II: Extract plasmids: X-3-2XI and XI-2-2XI, pHcsa9 and gRNA, stored at -20°C for future use. III: Perform single enzyme digestion with NotI on X-3-2XI and XI-2-2XI plasmids, confirm band sizes by agarose gel electrophoresis, and recover bands after enzyme digestion. IV: Shake-culture the following strains: xyl-cas9, Xyl, xyl-8XI, xyl-8XI-nfsI, xyl-8XI-ISU1, xyl-8XI-nfsI-ISU1. Detailed records of the main experiments:The basic principle of enzyme digestion and ligation: Enzyme digestion and ligation technology relies on the action of DNA restriction endonucleases. These enzymes can specifically recognise particular sites within DNA sequences and cleave the DNA at these sites, generating either sticky ends or blunt ends. By using restriction endonucleases, the target gene and vector DNA can be cut into linear molecules, which are then ligated together using DNA ligase to form a recombinant plasmid. Specific steps of enzyme digestion and ligation: Vector preparation: Select an appropriate vector, such as a plasmid, that contains an origin of replication, selection markers, and multiple cloning sites. Use restriction endonucleases to cut the vector, generating linearised vector DNA. Insert preparation: Similarly, restriction endonucleases are used to cut the target gene, generating a linearised fragment of the target gene DNA. Ligation reaction: Purify the digested vector and target gene fragment and use enzymes like T4 DNA ligase to ligate them together. The ligation reaction requires the appropriate reaction conditions, such as ATP, DTT, and Mg2+. Transformation and selection: Transform the ligation product into competent cells and select for successfully transformed clones using antibiotic resistance or other selection markers. Through these steps, the target gene can be stably inserted into the vector, creating a recombinant plasmid, which serves as the basis for subsequent gene expression, functional studies, and other applications. Part 5: I: After transferring and culturing the yeast, culture at 30 degrees Celsius until reaching an OD of 0.8-1.0 to prepare yeast-competent cells. II: Transform the pHcsa9 plasmid into xyl yeast using the LiAc method, and transform both the gRNA and the recovered two-copy fragments into xyl-cas9 yeast. III: Following heat shock transformation, recover by incubating for 1-2 hours, plate on YPD-Amp plates, and culture at 30 degrees Celsius overnight. Detailed records of the main experiments:The preparation steps for the yeast competent cells mainly include the following stages: Dip a sterile toothpick into an appropriate amount of yeast glycerol stock and streak a line on a YPD solid agar plate to isolate single colonies. Pick a single colony and inoculate it into a 3 mL YPD liquid culture tube, then incubate overnight at 30°C and 240 rpm on a shaker. Dilute the overnight culture 10-fold and measure the OD600, ensuring that the OD is more significant than 0.2. Inoculate a 250 mL conical flask containing 25 mL of 2×YPD medium with an initial OD600 of 0.2, and culture at 30°C and 240 rpm until the OD600 reaches 0.8 to 1.0. Centrifuge the culture at 20°C and 3000 rpm for 5 minutes to collect the cells, resuspend the cells in 25 mL of sterile water, and repeat the centrifugation step twice. Prepare a 42°C water bath and a 100°C metal bath. Add 1 mL of sterile water to resuspend the cells, transfer the cell suspension to a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 30 seconds, discard the supernatant, collect the cells, and obtain the yeast-competent cells. The prepared, competent cells should be aliquoted and stored at -80°C for long-term storage and use. These steps and considerations ensure the successful preparation of brewing yeast competent cells, providing a foundation for subsequent genetic operations and experiments. Part 6: I: Verify the xyl-8XI single clone yeast colony by colony PCR and confirm the target bands by agarose gel electrophoresis. II: Amplify the upstream and downstream homologous arms of the nfs1 gene and recover the fragments. III: Transform the recovered fragments and the pscm-nfs1 plasmid into the xyl-8XI strain and culture overnight at 30 degrees Celsius. Detailed records of the main experiments: S.cerevisiae competent cell transformation method using LiAc. Table 1 Reagents for LiAc conversion | | | | --- | --- | | Compositions | Volume (μL) | | PEG 3350（500 g/L） | 240 | | LiAc（1 M） | 36 | | Salmon sperm ssDNA（2 mg/L） | 50 | | DNA + H2O | 34 | First, place 2 mg/L salmon sperm DNA in a 100°C metal bath for 5 minutes. After 5 minutes, quickly remove it and put it on ice for later use. Add the following components sequentially to the centrifuge tube containing the collected competent cells, following the order and quantities as shown in Table 1. Perform a 42°C heat shock for 15 minutes, centrifuge at 12000 rpm for 30 seconds, discard the supernatant, add 1 mL YPD liquid medium, resuspend the cells, and seal with parafilm. Place it on a shaker, recover at 30°C and 240 rpm for 2 hours, then centrifuge at 3000 rpm for 4 minutes, discard 600 μL of the supernatant, resuspend, and spread 200 μL of the cell suspension evenly on a solid selection plate. Invert the plate and culture at 30°C for two days. Part 7: I: Verify the xyl-8XI-nfs1 single clone yeast colony by colony PCR and confirm the target bands by agarose gel electrophoresis. II: Amplify the upstream and downstream homologous arms of the ISU1 gene and recover the fragments. III: Transform the recovered fragments and the pscm-ISU1 plasmid into the xyl-8XI strain and culture overnight at 30 degrees Celsius. Part 8: I: Verify the xyl-8XI-ISU1 single clone yeast colony by colony PCR and confirm the target bands by agarose gel electrophoresis. II: Transform the recovered fragments and the pscm-ISU1 plasmid into the xyl-8XI-Nfs1 strain and culture overnight at 30 degrees Celsius. III: Inoculate and shake culture the 5 constructed strains overnight after the completion of the transformation. Part 9: I: Verify the xyl-8XI-ISU1 single clone yeast colony by colony PCR and confirm the target bands by agarose gel electrophoresis. II: Transform the recovered fragments and the pscm-ISU1 plasmid into the xyl-8XI-Nfs1 strain and culture overnight at 30 degrees Celsius. III: Shake the culture of the 5 constructed strains overnight after the completion of the transformation. Part 10: Ferment the constructed 4 strains along with the control strain in YPDX medium to assess the xylose metabolism of the strains.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one
iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{%
endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5351/description.png)
![](https://static.igem.wiki/teams/5351/description-c.png)
![](https://static.igem.wiki/teams/5351/description-b.png)
![](https://static.igem.wiki/teams/5351/description-a.png)
![](https://static.igem.wiki/teams/5351/ahaystack.png)
Engineering

CONTENT

* [Overview](#content-1)
* [part 1](#content-2)
* [part 2](#content-3)
* [part 3](#content-4)
* [part 4](#content-5)
* [part 5](#content-6)
* [part 6](#content-7)
* [Test](#content-8)
* [References](#content-9)

Overview

With the continuous demand for petrochemical fuels driven by economic development, the reserves of such
fuels on Earth are expected to be depleted in the future. Therefore, research on alternative energy sources
is becoming increasingly important [1-3]. Currently, scholars have explored numerous potential alternative
fuels, among which fuel ethanol stands out as one of the most promising substitutes for petrochemical fuels
due to its unique characteristics, such as economic viability, renewability, and environmental friendliness.
The production of fuel ethanol is derived from biomass, and the development of biomass energy globally has
become a significant component of the new energy landscape. Using lignocellulosic biomass as raw material to
produce second-generation fuel ethanol has become a mainstream direction for future development [4,5].
Therefore, it is essential to develop strains that can efficiently utilize both glucose and Xylose. This
product aims to construct a yeast strain capable of synthesizing ethanol from Xylose at a much higher
efficiency. To achieve this, we made four different strains by inserting 8 copies of the Xylose isomerase
gene, introducing a mutation that enhances Xylose metabolism, and knocking out a gene that inhibits Xylose
metabolism. Finally, we compared the Xylose metabolism efficiency of different strains to select the optimal
genetic combination.

![](https://static.igem.wiki/teams/5351/engineering1.png)

Fig. 1. The schematic diagram of Xylose metabolism

Part 1. pSCm-NFS1mu, BBa\_K5351001

###### Design 1:

The gRNA plasmid pSCm-NFS1 is designed for the yeast strain where the NFS1 gene is mutated. The critical
gene in the plasmid is the NFS1 gRNA, combined with the SNR52 promoter and SUP4 terminator. A mutation will
later be induced on the NFS1 gene in the yeast to promote the metabolism of Xylose. It is constructed by
connecting two N20 oligos and the gRNA backbone.

![](https://static.igem.wiki/teams/5351/engineering2.png)

Fig. 2. The plasmid map of pSCm-NFS1

###### Build 1:

The pSCm-N20 plasmid was cut by BsaI to obtain 5984 bp, 441 bp and 571 bp bands, and 5984 bp was recycled on
the gel as the backbone for backup.

![](https://static.igem.wiki/teams/5351/engineering3.png)

Fig. 3. Gel electrophoresis of enzyme cutting of pSCm-N20 backbone

The two synthesised N20 oligo, gRNA-492I-F and gRNA-492I-R, annealed and self-propelled to form a
double-stranded sequence using primer pairs, system: deionised water 35 µL, T4 ligase buffer 5 µL, primer
gRNA-492I-F 5 µL (20 µM), and gRNA-492I-R 5 µL (20 µM). Hold at 95 °C for 5 min, decrease 5-10 °C per
minute, hold at 16 °C for 10 min. Annealing products were diluted 10 times as fragments, and the gRNA
backbone was ligated to the above fragments with a T4 ligase kit at 16 °C. The plasmid was then transformed
into E. coli DH5α. LB plates containing a final concentration of 100 µg/mL ampicillin antibiotic were coated
and incubated overnight at 30 °C until there were transformants. Figure 4 shows the presence of single
colonies on the plate.

![](https://static.igem.wiki/teams/5351/engineering4.png)

Fig. 4. Transformation plate of pSCm-NFS1

A number of transformants were randomly picked and was verified by colony PCR using the primers
c-SCm-gRNA-F/gRNA-492I-R. The correct length of 288 bp was obtained. Figure 5 shows the band consistent with
the target size.

![](https://static.igem.wiki/teams/5351/engineering5.png)

Fig. 5. Gel electrophoresis validation of pSCm-NFS1

The transformants with the correct length were transferred to LB test tubes containing 100 µg/mL of
ampicillin antibiotic, cultured at 30 °C, 220 rpm overnight, and the plasmid was extracted.

The plasmid was sent for sequencing, and the primer for sequencing was c-SCm-gRNA-F, and 1 reaction was
measured. According to the results shown in Figure 6, the parts are successfully ligated, confirming the
successful construction of the pSCm- NFS1 plasmid.

![](https://static.igem.wiki/teams/5351/engineering6.png)

Fig. 6. Sequencing map of pSCm-NFS1

###### Learn 1:

The pSCm-NFS1 plasmid is constructed for the mutation of the NFS1 gene in the yeast using homologous
recombination to promote Xylose metabolism capability.

Part 2. pSCm-ISU1, BBa\_K5351004

###### Design 2:

The key gene in the pSCm-△ISU1 plasmid is ISU1 gRNA, with SNR52 promoter and SUP4 terminator. It is
constructed by connecting two N20 oligos and the gRNA backbone.

![](https://static.igem.wiki/teams/5351/engineering7.png)

Fig.7. The plasmid map of pSCm-△ISU1

###### Build 2:

The two synthesised N20 oligos, N20-ISU1-F and N20-ISU1-R, were first annealed and self-ligated to form a
double-stranded sequence. system: deionised water 35 µL, T4 ligase buffer 5 µL, primers N20-ISU1-F 5 µL (20
µM), N20-ISU1-R 5 µL (20 µM). Hold at 95 °C for 5 min, decrease the temperature by 5-10 °C per minute and
hold at 16 °C for 10 min. The annealed product was diluted 10-fold as a fragment, and the gRNA backbone was
chemically transformed to DH5α by ligating the above fragment with the T4 ligase kit at 16 °C in vitro. LB
plates containing a final concentration of 100 µg/mL ampicillin antibiotic were coated and cultured in an
incubator at 30 °C overnight until there were transformants. Figure 8 shows the presence of single colonies
on the plate.

![](https://static.igem.wiki/teams/5351/engineering8.png)

Fig. 8. Transformation plate of pSCm-△ISU1

A number of transformants were randomly picked and were verified by colony PCR using the primers
c-SCm-gRNA-F / N20-ISU1-R. The correct length of 288 bp was obtained. Figure 9 shows the band consistent
with the target size.

![](https://static.igem.wiki/teams/5351/engineering9.png)

Fig. 9. Gel electrophoresis validation of pSCm-△ISU1

The transformants with the correct length were transferred to LB test tubes containing 100 µg/mL of
ampicillin antibiotic, cultured at 30 °C, 220 rpm overnight, and the plasmid was extracted and sequenced.
The sequencing primer was c-SCm-gRNA-F, and 1 reaction was measured. According to the results shown in
Figure 10, the parts are successfully ligated, confirming the successful construction of the pSCm-△ISU1
plasmid.

![](https://static.igem.wiki/teams/5351/engineering10.png)

Fig. 10. Sequencing map of pSCm-△ISU1

###### Learn 2:

The pSCm-△ISU1 plasmid is constructed for the knockout of the ISU1 gene in the yeast using
homologous recombination. In order to promote the Xylose metabolism capability of yeast.

Part 3. X-3-XI, BBa\_K5351006

###### Design 3:

X-3-XI is constructed by integrating the PsXI gene into the X-3 site. A biotech company provides the
backbone plasmid of the X-3 integration site. The PsXI gene is joint by TEF1 promoter and ADH1 terminator.

![](https://static.igem.wiki/teams/5351/engineering11.png)

Fig. 11. The plasmid map of X-3-XI

###### Build 3:

The pHCas9 plasmid was used as a template, and primer pair TEF1p-F1/ TEF1p-XI-R1 amplified the TEF1 promoter
with a size of 430bp; the FDP-PsXI plasmid containing the PsXI gene was used as a template, and primer pair
XI-TEF1p-F1/ XI-ADH1t-R1 amplified the PsXI gene sequence with a size of 1,354bp. The brewer's yeast
colonies or genome as a template, primer pair ADH1t-XI-F1/ ADH1t-R1 amplified ADH1 terminator with a size of
214bp. Figure 12 shows the band consistent with the target size, indicating successful amplification.

![](https://static.igem.wiki/teams/5351/engineering12.png)

Fig. 12. Gel electrophoresis validation of gene amplification of TEF1 promoter, PsXI, and ADH1 terminator

The above three fragments were processed by overlap PCR using primer TEF1p-F1/ ADH1t-R1. The size of the
fragment was 1920bp. Figure 13 shows the band consistent with the target size, indicating the overlap PCR is
successful.

![](https://static.igem.wiki/teams/5351/engineering14.png)

Fig. 13. Overlap PCR results of TEF1-PsXI-ADH1

The 1920 bp fragment was recycled and ligated into the PsXI gene sequence, also using the primer TEF1p-F1/
Xho1. The 1920bp fragment was recycled using Sgs1+Xho1 double digestion and ligated into the X-3 locus
integration backbone plasmid. It was also double digested using Sgs1+Xho1. Figure 14 shows the band
consistent with the target size. The enzyme cutting is successful.

![](https://static.igem.wiki/teams/5351/engineering14.png)

Fig. 14. Results of enzyme cutting of X-3 backbone and TEF1-PsXI-ADH1

The plasmid is transformed into DH5α and cultivated on LB-Amp plates. Figure 15 shows the presence of single
colonies on the plate.

![](https://static.igem.wiki/teams/5351/engineering15.png)

Fig .15. Transformation plate of X-3-XI

Colonial PCR was carried out using the primers grna-HYG-baseplasmid-seq-7/ XI-ADH1t-R1. The
target band was 2065bp. Figure 16 shows the band consistent with the target size.

![](https://static.igem.wiki/teams/5351/engineering16.png)

Fig. 16. Gel electrophoresis validation of X-3-XI

Sequence using the primers grna-HYG-baseplasmid-seq-7 and h-x-3d-bb-r1. According to the results shown in
Figure 17, the construction of the X-3-XI plasmid is successful.

![](https://static.igem.wiki/teams/5351/engineering17.png)

Fig. 17. Sequencing map of X-3-XI

###### Learn 3:

X-3-XI plasmid is prepared for a second round of PsXI gene integration into the X-3-XI to produce a plasmid
with two copies of the PsXI gene, which would result in 8 copies of the gene in the yeast.

Part 4. XI-2-XI, BBa\_K5351008

###### Design 4:

PCR was used to obtain the backbone XI-2 and the target gene TEF1-PsXI-ADH1, followed by a link between the
XI-2 backbone and the target gene TEF1-PsXI-ADH1.

![](https://static.igem.wiki/teams/5351/engineering18.png)

Figure 18. The plasmid map of XI-2-XI

###### Build 4:

We constructed a plasmid XI-2-XI containing a single copy of the XI gene. We amplified and validated the
backbone XI-2 and the target gene TEF1-PsXI-ADH1. The results in Figure 19 showed matching band sizes,
indicating successful amplification. We ligated the XI-2 backbone and the target gene TEF1-PsXI-ADH1.

![](https://static.igem.wiki/teams/5351/engineering19.png)

Figure 19. Validation result of linear pX-3, linear pXI-2, and TEF1-PsXI-ADH1

We ligated the XI-2 backbone and the target gene TEF1-PsXI-ADH1 and transformed it into competent E.coli
DH5α. Figure 20 shows the results after culturing E. coli, where single colonies can be observed.

![](https://static.igem.wiki/teams/5351/engineering20.png)

Figure 20. XI-2-XI colonies

We performed colony PCR to validate the cultured colonies. Figure 21 displays the results of the colony PCR,
showing bands of approximately 2065 bp, consistent with the expected fragment size, validating our
successful transformation and plasmid construction.

![](https://static.igem.wiki/teams/5351/engineering21.png)

Figure 21. Validation of colony PCR of XI-2-PsXI

The colonies were also sent for sequencing. According to the results shown in Figure 22, the TEF1-PsXI-ADH1
gene was successfully ligated to the backbone without any apparent mutations, confirming the successful
construction of the XI-2-XI plasmid.

![](https://static.igem.wiki/teams/5351/engineering22.png)

Figure 22. Sequencing result of XI-2-XI

###### Learn 4:

Based on the construction of the X-3-XI plasmid, the newly constructed XI-2-XI plasmid has its integration
site modified to XI-2.

Part 5. X-3-2XI, BBa\_K5351007

###### Design 5:

A biotech company synthesized the target gene, XI. Obtain the promoter GAP, the coding gene PsXI, and the
terminator PsXI separately. Perform overlap PCR to connect these three fragments and finally ligate them to
the linearized vector.

![](https://static.igem.wiki/teams/5351/engineering23.png)

Figure 23. The plasmid map of X-3-2XI

###### Build 5:

We constructed a plasmid X-3-2XI containing two copies of the XI genes. Figure 24 shows the PCR validation
results for the promoter GAP, the coding gene PsXI, and the terminator CYC1, with band sizes matching the
expected lengths of 693 bp, 1350 bp, and 275 bp, respectively, indicating successful amplification.

![](https://static.igem.wiki/teams/5351/engineering24.png)

Figure 24. Amplification result of GAP pro, PsXI, and CYC1 ter

Overlap PCR was performed on these fragments. Figure 25 shows the results of the overlap PCR, with a band
size consistent with the expected length of 2245 bp, indicating successful synthesis of the target gene.

![](https://static.igem.wiki/teams/5351/engineering25.png)

Figure 25. Overlap PCR result of GAP-PsXI-CYC1

We amplified and validated the backbone X-3-XI and the target gene GAP-PsXI-CYC1. The results in Figure 26
showed matching band sizes, indicating successful amplification.

![](https://static.igem.wiki/teams/5351/engineering26.png)

Figure 26. Validation of X-3-XI, XI-2-XI, and GAP-PsXI-CYC1

We ligated the X-3-XI backbone and the target gene GAP-PsXI-CYC1 and transformed it into competent E.coli
DH5α. Figure 27 shows the results after culturing E. coli, where single colonies can be observed.

![](https://static.igem.wiki/teams/5351/engineering27.png)

Figure 27. X-3-2XI colonies

We performed colony PCR to validate the cultured colonies. Figure 28 displays the results of the colony PCR,
showing bands of approximately 904 bp, consistent with the expected fragment size, validating our successful
transformation and plasmid construction.

![](https://static.igem.wiki/teams/5351/engineering28.png)

Figure 28. Validation of colony PCR of X-3-2XI

The colonies were also sent for sequencing. According to the results shown in Figure 29, the GAP-PsXI-CYC1
gene was successfully ligated to the backbone without any apparent mutations, confirming the successful
construction of the X-3-2XI plasmid.

![](https://static.igem.wiki/teams/5351/engineering29.png)

Figure 29. Sequencing result of X-3-2XI

###### Learn 5:

Based on the construction of the X-3-XI plasmid, the newly constructed X-3-2XI plasmid obtained two copies
of the XI gene.

Part 6. XI-2-2XI, BBa\_K5351009

###### Design 6:

PCR was used to obtain the backbone XI-2-XI and the target gene GAP-PsXI-CYC1, followed by a link of the
XI-2-XI backbone and the target gene GAP-PsXI-CYC1.

![](https://static.igem.wiki/teams/5351/engineering30.png)

Figure 30. The plasmid map of XI-2-2XI

###### Build 6:

We constructed a plasmid XI-2-2XI containing two copies of the XI genes. We amplified and validated the
backbone XI-2-XI and the target gene GAP-PsXI-CYC1. The results in Figure 31 showed matching band sizes,
indicating successful amplification.

![](https://static.igem.wiki/teams/5351/engineering31.png)

Figure 31. Validation result of X-3-XI, XI-2-XI, and GAP-PsXI-CYC1

We ligated the XI-2-XI backbone and the target gene GAP-PsXI-CYC1 and transformed it into competent E.coli
DH5α. Figure 32 shows the results after culturing E. coli, where single colonies can be observed.

![](https://static.igem.wiki/teams/5351/engineering32.png)

Figure 32. XI-2-2XI colonies

We performed colony PCR to validate the cultured colonies. Figure 33 displays the results of the colony PCR,
showing bands of approximately 904 bp, consistent with the expected fragment size, validating our successful
transformation and plasmid construction.

![](https://static.igem.wiki/teams/5351/engineering33.jpg)

Figure 33. Validation of colony PCR of XI-2-2XI

The colonies were also sent for sequencing. According to the results shown in Figure 34, the GAP-PsXI-CYC1
gene was successfully ligated to the backbone without any apparent mutations, confirming the successful
construction of the XI-2-2XI plasmid.

![](https://static.igem.wiki/teams/5351/engineering34.png)

Figure 34. sequencing result of XI-2-2XI

###### Learn 6:

Based on the construction of the X-2-XI plasmid, the newly constructed XI-2-2XI plasmid obtained two copies
of the XI gene.

Test

###### A: Solid medium assay for determining strains' Xylose metabolism

The strains Xyl-8XI, Xyl-8XI-NFS1, Xyl-8XI-ΔISU1, Xyl-8XI-NFS1-ΔISU1, and Xyl-8XI-NFS1-ΔISU1 were evaluated
for their Xylose utilization capabilities, and their growth performances were compared. The Xylose plate
test results (Figure 35) revealed that the Xyl-8XI-NFS1 strain exhibited robust growth even under Xylose
dilutions of 10-12, surpassing the performance of Xyl-8XI, Xyl-8XI-ΔISU1, and Xyl-WT strains. This
observation suggests that the mutation of the NFS1 gene positively influenced Xylose metabolism.
Specifically, Xylose metabolic capacity was ranked as follows: XYL-8XI-NFS1 > XYL-8XI-NFs1-ΔISU1 > Xyl-8XI >
XYL-8XI-ΔISU1 > XYL-8XI-ΔISU1 > Xyl-WT. These results indicated that NFS1 gene mutation significantly
improved Xylose metabolic capacity, while ISU1 gene knockout had a specific effect on Xylose metabolism, but
it was not as significant as NFS1 mutation. Through this experiment, we have identified promising targets
for Xylose metabolism enhancement.

![](https://static.igem.wiki/teams/5351/engineering35.png)

Figure 35. Xylose plate assay for Xylose metabolism capability

###### B: Using HPLC to analyze the fermentation of strains

High-performance liquid chromatography (HPLC) testing will further quantitatively analyze the Xylose
metabolism to verify the actual performance of different strains in Xylose metabolism. The strains were
fermented in a YPDX medium to evaluate their actual Xylose metabolism efficiency. The results of HPLC will
further support the advantages of NFS1 mutant strains in Xylose metabolism, and these results provide new
ideas and theoretical basis for yeast strains to optimize Xylose utilization. From Figure 36, it is evident
that introducing Xylose isomerase into different parent strains resulted in enhanced Xylose metabolism
capabilities. Further validation of the NFS1 mutant strains' advantages in Xylose metabolism was achieved
through the actual Xylose metabolism performance in the fermentation broth. The strain with the fastest
Xylose utilization rate nearly depleted the Xylose in the medium within 40 hours. Additionally, observations
on strain growth revealed that the modified strains exhibited minimal impact on growth, with an earlier
onset of exponential growth. This rapid growth of the strains also positively influenced Xylose utilization.

![](https://static.igem.wiki/teams/5351/engineering36.png)

Figure 36. Comparison of Xylose metabolism and growth status of the modified strains (a: Xylose metabolism
profiles of different strains; b: Growth status comparison among different strains)

The ultimate goal of enhancing Xylose utilization in our engineered strains is for ethanol production.
Therefore, we also measured the ethanol content in the fermentation broth after fermentation to assess the
potential of our strains in the field of second-generation biofuel ethanol production. Experimental results,
as shown in Table 1, indicate a significant improvement in ethanol production capacity in the engineered
strains. Notably, our strain Xyl-8XI-nfs1 exhibited a 5.2-fold increase in ethanol production compared to
the parental strain, reaching 662.18 mg/L. This result further validates the success of our genetic
modifications to the strains.

###### Table 1. The ethanol production levels of different strains (48 h)

|  |  |
| --- | --- |
| Strains | Ethanol concentration (mg/L) |
| Xyl | 106.72 |
| Xyl-8XI | 298.74 |
| Xyl-8XI-nfs1 | 662.18 |
| Xyl-8XI-△ISU1 | 367.16 |
| Xyl-8XI-nfs1-△ISU1 | 307.10 |

###### Learn:

In our experiment, we successfully constructed a strain capable of utilizing Xylose and identified a
superior target gene. However, we utilized YPDX medium instead of glucose and Xylose derived from
lignocellulosic biomass hydrolysate. Therefore, in the future, we will need to ferment with actual
hydrolysate, which contains not only glucose and Xylose but also a significant amount of inhibitors
generated from lignin decomposition. This poses specific requirements for the stress tolerance of our
strains. We aim not only to enhance the strains' Xylose utilization capability but also to increase their
robustness in the hydrolysate, ultimately facilitating improved production of second-generation biofuel
ethanol.

References

[1] Li X, Wang Y, Li G, et al. Metabolic network remodelling enhances yeast's fitness on xylose using
aerobic
glycolysis[J]. Nature Catalysis, 2021, 4(9): 1-14.

[2] Zhu L, Li P, Sun T, et al. Overexpression of SFA1 in engineered Saccharomyces cerevisiae to increase
xylose utilization and ethanol production from different lignocellulose hydrolysates[J]. Bioresour Technol,
2020, 313: 123724.

[3] Shaobo Liang, Yinglei Han, Liqing Wei, et al. Production and characterization of bio-oil and bio-char
from
pyrolysis of potato peel wastes[J]. Biomass Conversion and Biorefinery, 2015, 5(3): 237-246.

[4] Matuštík Jan, Kočí Vladimír. Does renewable mean good for the climate? Biogenic carbon in climate impact
assessments of biomass utilization[J]. GCB Bioenergy, 2022, 14(4): 438-446.

[5] Meher L C, Sagar D V, Naik S N. Technical aspects of biodiesel production by transesterification—a
review[J]. Renewable and Sustainable Energy Reviews, 2006, 10(3): 248-268.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5351/description.png)
![](https://static.igem.wiki/teams/5351/description-c.png)
![](https://static.igem.wiki/teams/5351/description-b.png)
![](https://static.igem.wiki/teams/5351/description-a.png)
![](https://static.igem.wiki/teams/5351/ahaystack.png)
Experiment

CONTENT

* [Disposition of Culture](#content-1)
* [The Construction of Plasmid](#content-2)
* [Yeast Transformation](#content-3)
* [Functional Test](#content-4)

Disposition of Culture

###### 1.1.LB culture

Goal:
To create a functional and nutritional liquid environment for bacteria to grow. Activation, cultivation
for microorganisms.

###### Materials

* Yeast Extract
* Sodium chloride
* dd H2O
* Sterilized bottle, cap
* Lab Refrigerator
* Tryptone
* Autoclave
* Agar

###### Procedures

1)Create LB Medium (Used for maintaining and cultivating recombinant strains of E.coli).

2)Prepare 20g of premix LB broth powder (Tryptone: Yeast extract: Sodium chloride= 2:1:1)
(Note: 1.5% agar
powder was added to the solid culture medium).

3)Add into a sterilized bottle.

4)Add double distilled H2O to total volume of 1000ml.

5)Close the cap and autoclave it for 20 minutes at least.

6)Wait until it cools down to room temperature.

7)Store at 4℃ in the lab refrigerator.

###### 1.2.YPD/X culture

Goal:
To create a functional and nutritional environment for Saccharomyces cerevisiae to grow. Activation,
cultivation, and fermentation for Saccharomyces cerevisiae.

###### Materials

* Yeast Extract
* Glucose
* Tryptone
* Xylose
* Agar

###### Procedures

1)Dissolve 10 g yeast extract and 20 g tryptone in 900 mL water. Note: If preparing agar plates, add 20g
agar
at this point.

2)Sterilize the mixture under high pressure, typically maintaining at 115°C for 15 minutes.

3)Prepare 100ml of 20% glucose solution.

4)Add glucose separately after sterilization. Note: If adding xylose, sterilize individually and add the
calculated amount to achieve the desired concentration.

The Construction of Plasmid

###### 2.1.Construction of pSCm-NFS1mu

###### A.Obtaining the gene fragment from pSCm-N20

Goal:
To obtain the gene fragment (length of 5984 bp) from pSCm-N20 for later recombination.

###### Materials

* Bsal Enzyme
* pSCm-N20

###### Procedures

Use Enzyme Bsal to cut pSCm-N20 and get 5984 bp、441 bp, and 571 bp.

###### B.Agarose gel construction, deployment and recycle

Goal:
To produce a functional and well-shaped gel for electrophoresis

###### Materials

* pipettor
* Casting Tray
* Microwave
* 1x TAE
* Agarose M
* 10000x Nucleic acid gel stain
* Erlenmeyer flask

###### Procedures

1)Dissolve 0.3g Agarose in 30mL Erlenmeyer flask

2)Add TAE (Running buffer)

3)Heat the material with a microwave

4)Melt the mixture at 30 sec intervals

5)See whether bubbles are forming if successfully mixed

6)Add 3μl nucleic acid gel stain (10000x)

7)Let it cool for 5~10 minutes

8)Pour the agarose mixture into the casting tray

9)Place the appropriate healthy comb to create the wells

10)Wait to solidify ~30 minutes

11)Remove the comb and place the gel in the gel box

###### C.Agarose gel electrophoresis

Goal:
To verify the DNA bands’ lengths of the plasmid extracted and recollect the 5984bp later.

###### Materials

* Sample
* Marker
* Prepared gel (from the previous step)
* 10x Loading buffer (dye)
* Horizontal gel electrophoresis

###### Procedures

1)Extract 50µl of every sample; add 5µl of loading buffer (10x) to each.

2)Place the gel in the horizontal electrophoresis system

3)Extract 50µl of the mixed solution containing the sample and mix them till their colors are
almost uniform

4)Add the marker to the first well and load 50µl of each sample into the wells

5)The procedure runs at 160V for 30min

6)Afterwards, take out the gel from the device and proceed to the following steps

###### D.DNA Gel Extraction

Goal:
To recover 5984 bp in agarose gel.

###### Materials

* agarose gel with 5984 bp
* clean scalpel
* 1.5mLEppendorf (EP) tube
* pipettes and sterilized pipette heads
* hot water bath
* buffer B2
* purification column and collection tube
* centrifuge
* wash solution containing pure ethanol

###### Procedures

1)Cut the slice of gel containing 5984 bp, cutting off as much unneeded gel as possible, and
place it in a 1.5
mL Eppendorf (EP) tube.

2)Add 500 microliters of buffer B2 and put the tube into a 50°C hot water bath until the gel
has completely
melted.

3)Transfer the solution containing melted gel into a purification column that's in a
collection tube and
perform centrifugation at 8000 g for 30 seconds.

4)Empty the collection tube, put the purification column back in, and add 500 microliters of
wash solution
containing pure ethanol. Centrifugation at 9000 g was performed for 30 seconds, and the collection tube was
emptied again.

5)Repeat above.

6)Perform centrifugation one more time at 9000 g for 1 minute, then open the cap of the tube
and let it sit
for one minute to allow the ethanol to evaporate.

7)Transfer the purification column to a 1.5 mL EP tube. Add 20 microliters of elution buffer
at the center of
the purification column.

8)Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 9000 g for 1
minute.

9)Discard the purification column. Store DNA in the EP tube at 4°C

###### E.Transferring the recombined fragment into DH5α

Goal:
Transform the constructed plasmid into Escherichia coli for validation and amplification.

###### Materials

* Plasmid after ligation
E. coli in a competent state

###### Procedures

1) Ligated samples are added to E.Coli samples

2) Placed on ice for 20 minutes.

3) Heat at 42 ℃ for 45 seconds, then immediately put on ice for 2-3 minutes.

4) Add 900μL LB Agar solution and incubate for 30 minutes

5) Centrifuge at 5000xg for 3 minutes, extract 900μL of supernatant, then discard

6) Apply the end product to agar plates and incubate for 12-16 h at 37 degrees Celsius.

###### F.Colony PCR Validation

Goal:
Verify the transformants and inoculate them to grow with the target gRNA.

###### Materials

* PCR tube
* 2xmix
* C-SCm-gRNA-F
* N20-ISU1-R
* ddH2O
* LB cultural base
* Bacterial colonies
* PCR machine

###### Procedures

1)Prepare the system (20 μl: 10 μl ClonExpress Mix, 1 μl primer-F, 1 μl primer-R, 8ul ddH2O)

2)Pick a single colony with a 2.5 μl sterile pipette tip and perform aspiration and dispensing
in the system.

3)Insert the mixture into a PCR thermal cycler and design the PCR program according to the
manufacturer's
instructions.

4)Perform agarose gel electrophoresis, and the presence of the correct bands indicates the
successful
construction of the plasmid pSCm-NFS1mu.

###### G.Extracting the plasmid

Goal:
To obtain the plasmid pSCm-NFS1mu from DH5α

###### Materials

* Centrifuge tube
* Vortex
* Solution I
* Solution II
* Solution III
* Pipette
* Anhydrous ethanol
* Rinse solution
* Absorbent column
* Elution buffer
* Centrifuge

###### Procedures

1)Take 2 mL of bacterial culture and add it to a 2 mL centrifuge tube, centrifuge at 12000 rpm
for 1 min and
aspirate as much supernatant as possible.

2)Add 2 mL of bacterial culture to the same tube and centrifuge at 12000 rpm for 1 min; remove
as much
supernatant as possible.

3)Add 250 μL of Solution I to the centrifuge tube with the bacterial sediment and use a vortex
shaker to
suspend the bacterial cell sediment thoroughly.

4)Add 250μL of Solution II to the centrifuge tube and gently turn the tube up and down 6-8
times to fully lyse
the bacteria.

5)Add 350μL of Solution III to the centrifuge tube and immediately mix well by gently turning
the tube up and
down 6-8 times. The centrifuge was at 12000 rpm for 10 min, and the supernatant was carefully transferred to
another clean centrifuge tube using a pipette.

6)Take the supernatant from the previous step, add 0.35 times the volume of anhydrous ethanol
and mix
thoroughly.

7)Add the supernatant/mixing solution from the previous step into the adsorbent column
(adsorbent column into
the collection tube), let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, pour out
the waste liquid in the collection tube, and put the adsorbent column back into the collection tube.

8)Add 750 μL of rinse solution to the column, centrifuge at 12000 rpm for 1 min, discard the
waste solution,
and put the column back into the collection tube.

9)Add 700 μL of rinse solution to the column, centrifuge at 12000 rpm for 1 min, discard the
waste solution,
and place the column in a collection tube.

10)Centrifuge at 12000 rpm for 2 min and leave the column open at room temperature for a few
minutes.

11)Place the column into a clean centrifuge tube, add 40 drops of eluent to the centre of the
adsorbent
membrane, leave it at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min.

###### 2.2.Construction of pSCm-ΔISU1

###### A. Construction of pSCm-ΔISU1

Goal:
Obtained the pSCm vector backbone and the target gene ΔISU1.

Materials:
Designed primers specific to pSCm-ΔISU1 construction, keeping other materials consistent with the
previous procedure.

###### 2.3.Construction of X-3-X

###### A. Construction of X-3-X

Goal:
Constructing a plasmid X-3-XI containing a single copy of the XI gene. The promoter TEF1, coding gene
PsXI, and terminator are 430 bp, 1354 bp, and 214 bp in length, respectively.

Materials:
Designed primers specific to X-3-X construction, keeping other materials consistent with the
previous procedure.

###### 2.4.Construction of XI-2-XI

###### A. Construction of XI-2-XI

Goal:
Construct a plasmid XI-2-XI containing a single copy of the XI gene.

Materials:
Designed primers specific to X-3-X construction, keeping other materials consistent with the
previous procedure.

###### 2.5.Construction of X-3-2XI

###### A. Construction of X-3-2XI

Goal:
Construct a plasmid X-3-2XI containing two copies of the XI gene. The lengths of the PCR products for
the GAP promoter, coding gene PsXI, and terminator gene CYC1 are 693 bp, 1350 bp, and 275 bp, respectively.
Perform overlap PCR on these fragments, resulting in a band length of 2245 bp. Perform ligation using
Mssl+Xhol double enzyme digestion. Ligated the X-3-XI backbone and the target gene GAP-PsXI-CYC1 and
transformed it into competent E.coli DH5α.

Materials:
Designed primers specific to X-3-2XI construction, keeping other materials consistent with the
previous procedure.

###### 2.6.Construction of XI-2-2XI

###### A. Construction of XI-2-2XI

Goal:
Constructing a plasmid XI-2-2XI containing two copies of the XI gene. Perform ligation using SgsI and
XhoI double enzyme digestion.

Materials:
Designed primers specific to XI-2-2XI construction, keeping other materials consistent with the
previous procedure.

Yeast Transformation

###### 3.1.xyl-8XI

We transferred the pHCas9-Nourse plasmid into the xyl-WT strain and screened the YPD-Nourse
resistance plate
to obtain xyl-Cas9 positive transformers. Then the X-3-XI-2-gRNA-HYG plasmid and the target gene fragment
containing 2 copies of the PsXI expression frame were transferred into the xyl-Cas9 strain, and the positive
invert was screened on the YPD-Nourse-HYG resistance plate. PsXI integration was then verified by colony PCR
with primers GAP-XI-F1 / h-x-3d-bb-r1 and GAP-XI-F1 / h-xi-2d-bb-r1. We successfully integrated 8 copies of
PsXI genes and named the strain xyl-8XI.

###### A. Preparation of S. cerevisiae competent cells.

Dip a sterile toothpick into a suitable amount of yeast liquid culture and streak it on a YPD solid agar plate
to isolate single colonies. Pick a single colony and inoculate it into a 3 mL YPD liquid culture tube, then
culture overnight at 30°C and 240 rpm on a shaker. Dilute the overnight culture 10 times and measure OD600,
ensuring that OD600 is more significant than 0.2. Inoculate a 250 mL conical flask containing 25 mL of 2×YPD
medium with an initial OD600 of 0.2, and culture at 30°C and 240 rpm until OD600 reaches 0.8 to 1.0.
Centrifuge the culture at 20°C and 3000 rpm for 5 minutes, collect the cells, resuspend them in 25 mL of
sterile water, and repeat the centrifugation step twice. Open a 42°C water bath and a 100°C metal bath.
Resuspend the cells in 1 mL of sterile water, transfer the cell suspension to a 1.5 mL centrifuge tube,
centrifuge at 12000 rpm for 30 seconds, discard the supernatant, collect the cells, and obtain competent yeast
cells.

###### B. S. cerevisiae competent cell transformation method using LiAc.

###### Table 1. Reagents for LiAc conversion

|  |  |
| --- | --- |
| Compositions Volume (μL) | Volume (μL) |
| PEG 3350（500 g/L） | 240 |
| LiAc（1 M） | 36 |
| Salmon sperm ssDNA（2 mg/L） | 50 |
| DNA + H2O | 34 |

First, place 2 mg/L salmon sperm DNA in a 100°C metal bath for 5 minutes. After 5 minutes, quickly remove it
and put it on ice for later use.
Add the following components sequentially to the centrifuge tube containing the collected competent cells,
following the order and quantities as shown in Table 1.

Perform a 42°C heat shock for 15 minutes, centrifuge at 12000 rpm for 30 seconds, discard the supernatant, add
1 mL YPD liquid medium, resuspend the cells, and seal with parafilm. Place it on a shaker, recover at 30°C and
240 rpm for 2 hours, then centrifuge at 3000 rpm for 4 minutes, discard 600 μL of the supernatant, resuspend,
and spread 200 μL of the cell suspension evenly on a solid selection plate. Invert the plate and culture at
30°C for two days.

###### 3.2.xyl-8XI-NFS1

After constructing strain xyl-8XI, we further mutated the nfs1 gene, mutated Ile at site 492 to Asn, and
constructed strain XYL-8XI-NFS1. We introduced PGMC-GRNA-NFS1MU and the amplified homologous arm fragment into
the prepared xyl-8XI receptive cells and cultured them at 30 °C. Subsequently, colony PCR was performed with
primer NFS1 (1513-1535) -F/ NFS1-DN (2044-2072) -R. We successfully integrated 8 copies of the PsXI gene, and
the Ile mutation at site 492 of the NFS1 gene was changed to Asn, and the resulting strain was named
xyl-8XI-nfs1. The preparation of competent cells and the transformation method are consistent with the above.

###### 3.3.xyl-8XI-ΔISU1

After the construction of strain xyl-8XI, we further performed gene knockout on the ISU1 gene to construct
strain xyl-8XI-ΔISU1. We introduced pSCm-gRNA-ΔISU1 and the amplified homologous arm fragment into the
prepared xyl-8XI receptive cells and cultured them at 30 °C. Colony PCR was performed with primer jd-ISU1
(282-308) -F/ jd-ISU1 (1209-1232) -R, and the final strain was named xyl-8XI-ΔISU1. We successfully integrated
8 copies of the PsXI gene and knocked out the ISU1 gene, and the resulting strain was named xyl-8XI-ΔISU1. The
preparation of competent cells and the transformation method are consistent with the above.

###### 3.4.xyl-8XI-nfs1-ΔISU1

After constructing strain xyl-8XI-nfs1, we further performed gene knockout on the ISU1 gene to construct
strain XYL-8XI-NFS1-ΔISU1. We introduced pSCm-ΔISU1 and homologous arm fragments into xyl-8XI-nfs1 receptor
cells and cultured them at 30 °C; Monoclonal colonies were selected for colony PCR verification using primer
jd-ISU1 (282-308) -F/ jd-ISU1 (1209-1232) -R. We successfully integrated 8 copies of PsXI genes. The Ile at
site 492 of the NFS1 gene was mutated into Asn, and the ISU1 gene was knocked out. The final strain was named
wx-xyl-8XI-nfs1-ΔISU1. The preparation of competent cells and the transformation method are consistent with
the above.

Functional Test

###### 4.1.Solid medium assay for determining strains' xylose metabolism

Goal:
Inoculate the 5 stains to test the xylose-consuming ability.

###### Procedures

1)Primary: Pick one single colony and transfer it to a 3 mL YPDX test tube; add prokaryotic
antibiotics,
ampicillin and kanamycin to prevent infection of prokaryotic organisms in the test tube. Incubate for 24 h at
30℃, 240 rpm, and the OD600 should reach more than 10 (Note: Unless otherwise specified, each experiment
consists of three replicates).

2)Secondary: Take 200 µL of primary seed bacterial solution and connect it to a fresh 3 mL
YPD10X tube,
incubate at 30℃, 240 rpm for 24 h. The culture was then incubated at 30℃ for 24 h.

3)Dispense fermentation medium YP40X: Dispense 5 mL/tube into sterile anaerobic tubes.

4)Calculate the inoculum: take the secondary seed broth and dilute it 20 times, measure the
OD600 by
spectrophotometer, and calculate the inoculum with the amount of 0.5g/L DCW; the calculation formula is:
Inoculum volume(mL)=((Fermentation volume V(mL))/2)/(0.63×Seed cultureOD600)=0.5gDCW/L.

5)Inoculation: Take the calculated inoculum amount using the formula above of to the sterile
1.5 mL
specification of the centrifuge tube.

6)1,2000 rpm centrifugation for 3 minutes.

7)Discard the supernatant wash with sterile water twice

8)1,2000 rpm centrifugation for 3 min, discard the supernatant.

9)Pipette 200 µL of culture medium from the above dispensed anaerobic tube into a 1.5 mL
centrifuge tube,
blowing it to make the yeasts float, and then transfer all of them back to the anaerobic tube and tighten the
cap

10)Place the above inoculated anaerobic tube at 30℃, 240 rpm shaker for incubation and record
the time.

###### 4.2.Using HPLC to analyze the fermentation of strains

Goal:
High-performance liquid chromatography (HPLC) testing will further quantitatively analyze the xylose
metabolism to verify the actual performance of different strains in xylose metabolism. The strains were
fermented in a YPDX medium to evaluate their actual xylose metabolism efficiency

###### Procedures

1）The cellular biomass of the bacterium was detected by spectrophotometer, adjusting the
wavelength to 600 nm,
calibrating the zero with deionized water, and diluting the sample of bacterial liquid by 10-20 times to
ensure that the OD600 of the spectrophotometric reading was in the range of 0 to 1.

2）Sample the fermentation broth every 8 hours by taking 200 µL of the culture into a 1.5 mL
centrifuge tube.
Use 100 µL for measuring OD600 and the remaining sample for HPLC analysis of xylose and ethanol until the end
of fermentation.

3）The concentrations of xylose and ethanol were measured using an HPLC system (Waters e2695)
equipped with an
Aminex HPX-87H ion exchange column (300 × 7.8 mm; Bio-Rad) at 35°C and a Waters 2414 refractive index
detector. Sulfuric acid (5 mM) served as the mobile phase with a flow rate of 0.6 ml min. The biological
company conducted the specific testing.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5351/description.png)
![](https://static.igem.wiki/teams/5351/description-c.png)
![](https://static.igem.wiki/teams/5351/description-b.png)
![](https://static.igem.wiki/teams/5351/description-a.png)
![](https://static.igem.wiki/teams/5351/ahaystack.png)
Results

CONTENT

* [Overview](#content-1)
* [Construction of plasmids](#content-2)
* [Yeast transformation](#content-3)
* [Functional Test](#content-4)
* [Discussion](#content-5)
* [References](#content-6)

Overview

With the continuous demand for petrochemical fuels driven by economic development, the reserves of such
fuels
on Earth are expected to be depleted in the future. Therefore, research on alternative energy sources is
becoming increasingly important [1-3]. Currently, scholars have explored numerous potential alternative
fuels,
among which fuel ethanol stands out as one of the most promising substitutes for petrochemical fuels due to
its unique characteristics, such as economic viability, renewability, and environmental friendliness. The
production of fuel ethanol is derived from biomass, and the development of biomass energy globally has
become
a significant component of the new energy landscape. Using lignocellulosic biomass as raw material to
produce
second-generation fuel ethanol has become a mainstream direction for future development [4,5]. Therefore, it
is essential to develop strains that can efficiently utilize both glucose and xylose. This product aims to
construct a yeast strain capable of synthesizing ethanol from xylose much more efficiently. To achieve this,
we made four different strains by inserting 8 copies of the xylose isomerase gene, introducing a mutation
that
enhances xylose metabolism, and knocking out a gene that inhibits xylose metabolism. Finally, we compared
the
xylose metabolism efficiency of different strains to select the optimal genetic combination.

Construction of plasmids

###### a) pSCm-NFS1mu

We constructed pSCm-NFS1mu by ligating the pSCm backbone and the target gene NFS1. The pSCm
was digested to
obtain a linear backbone with a length of 5984 bp. Figure. 1 indicates the band is consistent with the
results.

![](https://static.igem.wiki/teams/5351/result-1.png)

Figure 1. The gel electrophoresis validation of the linear pSCM-N20 plasmid backbone

These backbones were ligated with the target gene NFS1 and transformed into competent E.coli DH5α. Figure.
2a
shows the presence of single colonies on the plate.
Colony PCR was performed to validate the colonies. Figure. 2b displays the results of colony PCR, showing
bands of 288 bp, consistent with the expected length, thus validating our successful plasmid construction.

The colonies were also sent for sequencing. According to the results shown in the figure. 2c, the NFS1 gene
was successfully ligated to the backbone without any apparent mutations, confirming the successful
construction of the pSCm-NFS1 plasmid.

![](https://static.igem.wiki/teams/5351/result2.png)

Figure 2. The Monoclonal antibody validation and sequencing of pSCm-NFS1 (DH5α) (a: pSCm-NFS1 colonies; b:
the
gel electrophoresis validation of colony PCR of pSCm-NFS1; c: sequencing result of pSCm-NFS1)

###### b) pSCm-ΔISU1

We generated pSCm-ΔISU1 through the ligation of the pSCm backbone with the target gene ΔISU1, followed by
transformation into competent E. coli DH5α. Figure. 3a illustrates the presence of individual colonies on
the
plate.

A colony PCR was conducted to validate the colonies. The results are depicted in Figure. 3b exhibits bands
of
288 bp, aligning with the expected length, verifying the successful construction of the plasmid.
Furthermore, sequencing was performed on the colonies. As demonstrated in Figure. 3c, the ΔISU1 gene was
seamlessly ligated to the backbone without any detectable mutations, confirming the accurate construction of
the pSCm-ΔISU1 plasmid.

![](https://static.igem.wiki/teams/5351/result3.png)

Figure 3. The Monoclonal antibody validation and sequencing of pSCm-ΔISU1 (DH5α) (a: pSCm-ΔISU1 colonies; b:
the gel electrophoresis validation of colony PCR of pSCm-ΔISU1 colonies; c: sequencing result of pSCm-ΔISU1)

###### c) X-3-XI

We constructed a plasmid X-3-XI containing a single copy of the XI gene. Figure. 4 shows the PCR validation
results for the promoter TEF1, the coding gene PsXI, and the terminator ADH1, with band sizes matching the
expected lengths of 430 bp, 1354 bp, and 214 bp, respectively, indicating successful amplification.

![](https://static.igem.wiki/teams/5351/result4.png)

Figure 4. Amplification result of TEF1 pro, PsXI, and ADH1 ter

Overlap PCR was performed on these fragments. Figure. 5 shows the results of the overlap PCR, with a band
size
consistent with the expected length of 1920 bp, indicating successful synthesis of the target gene.

![](https://static.igem.wiki/teams/5351/result5.png)

Figure 5. Overlap PCR result of TEF1-PsXI-ADH1

We amplified and validated the backbone X-3 and the target gene TEF1-PsXI-ADH1. The results in Figure. 6
showed matching band sizes, indicating successful amplification.

![](https://static.igem.wiki/teams/5351/result6.png)

Figure 6. Validation of linear pX-3, linear pXI-2, and TEF1-PsXI-ADH1

We ligated the X-3 backbone and the target gene TEF1-PsXI-ADH1 and transformed it into competent E.coli
DH5α.
Figure. 7a shows the results after culturing E. coli, where single colonies can be observed.

We performed colony PCR to validate the cultured colonies and Figure. 7b displays the results of the colony
PCR, showing bands of approximately 2065 bp, consistent with the expected fragment size, validating our
successful transformation and plasmid construction.

The colonies were also sent for sequencing. According to the results shown in the figure. 7c, the
TEF1-PsXI-ADH1 gene was successfully ligated to the backbone without any apparent mutations, confirming the
successful construction of the X-3-XI plasmid.

![](https://static.igem.wiki/teams/5351/result7.png)

Figure 7. The Monoclonal antibody validation and sequencing of X-3-XI (DH5α) (a: X-3-XI colonies; b: the gel
electrophoresis validation of colony PCR of X-3-2XI colonies; c: sequencing result of X-3-2XI)

###### d) XI-2-XI

We constructed a plasmid XI-2-XI containing a single copy of the XI gene. We amplified and validated the
backbone XI-2 and the target gene TEF1-PsXI-ADH1. The results in Figure. 8 showed matching band sizes,
indicating successful amplification.

![](https://static.igem.wiki/teams/5351/result8.png)

Figure 8. Validation result of linear pX-3, linear pXI-2, and TEF1-PsXI-ADH1

We ligated the XI-2 backbone and the target gene TEF1-PsXI-ADH1 and transformed it into competent E.coli
DH5α.
Figure. 9a shows the results after culturing E. coli, where single colonies can be observed.

We performed colony PCR to validate the cultured colonies and Figure. 9b displays the results of the colony
PCR, showing bands of approximately 2065 bp, consistent with the expected fragment size, validating our
successful transformation and plasmid construction.

The colonies were also sent for sequencing. According to the results shown in the figure. 9c, the
TEF1-PsXI-ADH1 gene was successfully ligated to the backbone without any apparent mutations, confirming the
successful construction of the XI-2-XI plasmid.

![](https://static.igem.wiki/teams/5351/result9.png)

Figure 9. The Monoclonal antibody validation and sequencing of XI-2-XI (DH5α) (a: XI-2-XI colonies; b:
Validation of colony PCR of XI-2-PsXI; c: sequencing result of XI-2-XI)

###### e) X-3-2XI

We constructed a plasmid X-3-2XI containing two copies of the XI genes. Figure. 10 shows the PCR validation
results for the promoter GAP, the coding gene PsXI, and the terminator CYC1, with band sizes matching the
expected lengths of 693 bp, 1350 bp, and 275 bp, respectively, indicating successful amplification.

![](https://static.igem.wiki/teams/5351/result10.png)

Figure 10. Amplification result of GAP pro, PsXI, and CYC1 ter

Overlap PCR was performed on these fragments, and Figure. 11 shows the results of the overlap PCR, with a
band
size consistent with the expected length of 2245 bp, indicating successful synthesis of the target gene.

![](https://static.igem.wiki/teams/5351/result11.png)

Figure 11. Overlap PCR result of GAP-PsXI-CYC1

We amplified and validated the backbone X-3-XI and the target gene GAP-PsXI-CYC1. The results are in Figure.
12 showed matching band sizes, indicating successful amplification.

![](https://static.igem.wiki/teams/5351/result12.png)

Figure 12. validation of X-3-XI, XI-2-XI, and GAP-PsXI-CYC1

We ligated the X-3-XI backbone and the target gene GAP-PsXI-CYC1 and transformed it into competent E.coli
DH5α. Figure. 13a shows the results after culturing E. coli, where single colonies can be observed.

We performed colony PCR to validate the cultured colonies and Figure. 13b displays the results of the colony
PCR, showing bands of approximately 904 bp, consistent with the expected fragment size, validating our
successful transformation and plasmid construction.

The colonies were also sent for sequencing. According to the results shown in the figure. 13c, the
GAP-PsXI-CYC1 gene was successfully ligated to the backbone without any apparent mutations, confirming the
successful construction of the X-3-2XI plasmid.

![](https://static.igem.wiki/teams/5351/result13.png)

Figure 13. The Monoclonal antibody validation and sequencing of X-3-2XI (DH5α) (a: X-3-2XI colonies; b:
Validation of colony PCR of X-3-2XI; c: sequencing result of X-3-2XI)

###### f) XI-2-2XI

We constructed a plasmid XI-2-2XI containing two copies of the XI genes. We amplified and validated the
backbone XI-2-XI and the target gene GAP-PsXI-CYC1. The results are in Figure. 14 showed matching band
sizes,
indicating successful amplification.

![](https://static.igem.wiki/teams/5351/result14.png)

Figure 14. validation result of X-3-XI, XI-2-XI, and GAP-PsXI-CYC1

We ligated the XI-2-XI backbone and the target gene GAP-PsXI-CYC1 and transformed it into competent E.coli
DH5α. Figure. 15a shows the results after culturing E. coli, where single colonies can be observed.

We performed colony PCR to validate the cultured colonies and Figure. 15b displays the results of the colony
PCR, showing bands of approximately 904 bp, consistent with the expected fragment size, validating our
successful transformation and plasmid construction.

The colonies were also sent for sequencing. According to the results shown in the figure. 15c, the
GAP-PsXI-CYC1 gene was successfully ligated to the backbone without any apparent mutations, confirming the
successful construction of the XI-2-2XI plasmid.

![](https://static.igem.wiki/teams/5351/result15.png)

Figure 15. The Monoclonal antibody validation and sequencing of XI-2-2XI (DH5α) (a: XI-2-2XI colonies; b:
Validation of colony PCR of XI-2-2XI; c: sequencing result of XI-2-2XI)

Yeast transformation

###### a) xyl-8XI

First, we transferred the pHCas9-Nourse plasmid into the xyl-WT strain and screened the YPD-Nourse
resistance
plate to obtain xyl-Cas9 positive transformers. Then the X-3-XI-2-gRNA-HYG plasmid and the target gene
fragment containing 2 copies of the PsXI expression frame were transferred into the xyl-Cas9 strain, and the
positive invert was screened on the YPD-Nourse-HYG resistance plate. PsXI integration was then verified by
colony PCR with primers GAP-XI-F1\h-x-3d-bb-r1 and GAP-XI-F1\h-xi-2d-bb-r1. Agarose gel electrophoresis
results (Figure. 16) showed that the target band expanded 900 bp bands, as expected, indicating that we
successfully integrated 8 copies of PsXI genes and named the strain xyl-8XI.

![](https://static.igem.wiki/teams/5351/result16.png)

Figure 16. PCR and colony map of xyl-8XI

###### b) xyl-8XI-nfs1

After constructing strain xyl-8XI, we further mutated the nfs1 gene, mutated Ile at site 492 to Asn, and
constructed strain XYL-8XI-NFS1. We introduced PGMC-GRNA-NFS1MU and the amplified homologous arm fragment
into
the prepared xyl-8XI receptive cells and cultured them at 30 °C. Subsequently, colony PCR was performed with
primer NFS1 (1513-1535) -F/ NFS1-DN (2044-2072) -R. The result of agar-gel electrophoresis (Figure. 17)
showed
that the target band was amplified by about 495 bp, as expected, indicating that we successfully integrated
8
copies of the PsXI gene, and the Ile mutation at site 492 of the NFS1 gene was changed to Asn, and the
resulting strain was named xyl-8XI-nfs1.

![](https://static.igem.wiki/teams/5351/result17.png)

Figure 17. PCR validation diagram and colony diagram of xyl-8XI-nfs1

###### c) xyl-8XI-ΔISU1

After the construction of strain xyl-8XI, we further performed gene knockout on the ISU1 gene to construct
strain XYL-8XI-ΔISU1. We introduced pSCm-gRNA-ΔISU1 and the amplified homologous arm fragment into the
prepared xyl-8XI receptive cells and cultured them at 30 °C. Colony PCR was performed with primer jd-ISU1
(282-308) -F/ jd-ISU1 (1209-1232) -R, and the final strain was named xyl-8XI-ΔISU1. The results of agar-gel
electrophoresis (Figure. 18) showed that the target band was amplified by about 451bp, as expected,
indicating
that we successfully integrated 8 copies of the PsXI gene and knocked out ISU1 gene, and the resulting
strain
was named xyl-8XI-ΔISU1.

![](https://static.igem.wiki/teams/5351/resul18.png)

Figure 18. PCR and colony map of xyl-8XI-ΔISU1

###### d) xyl-8XI-nfs1-ΔISU1

After constructing strain xyl-8XI-nfs1, we further performed gene knockout on the ISU1 gene to construct
strain XYL-8XI-NFS1-ΔISU1. We introduced pSCm-ΔISU1 and homologous arm fragments into xyl-8XI-nfs1 receptor
cells and cultured them at 30 °C; Monoclonal colonies were selected for colony PCR verification using primer
jd-ISU1 (282-308) -F/jd-ISU1 (1209-1232) -R. Results of agar-gel electrophoresis (Figure. 19) showed that
the
target band was amplified by about 451bp, as expected, indicating that we successfully integrated 8 copies
of
PsXI genes. The Ile at site 492 of the NFS1 gene was mutated into Asn, and the ISU1 gene was knocked out.
The
final strain was named wx-xyl-8XI-nfs1-ΔISU1.

![](https://static.igem.wiki/teams/5351/result19.png)

Figure 19. Colony PCR and colony map of xyl-8XI-nfs1-ΔISU1

Functional Test

###### a) Solid medium assay for determining strains' xylose metabolism.

The strains Xyl-8XI, Xyl-8XI-NFS1, Xyl-8XI-ΔISU1, Xyl-8XI-NFS1-ΔISU1, and XYL-8XI-NFS1-ΔISU1 were evaluated
for their xylose utilization capabilities, and their growth performances were compared. The xylose plate
test
results (Figure. 20) revealed that the XYL-8XI-NFS1 strain exhibited robust growth even under xylose
dilutions
of 10-12, surpassing the performance of xyl-8XI, Xyl-8XI-ΔISU1, and xyl-WT strains.

This observation suggests that the mutation of the NFS1 gene positively influenced xylose metabolism.
Specifically, xylose metabolic capacity was ranked as follows:
XYL-8XI-NFS1>XYL-8XI-NFs1-ΔISU1>xyl-8XI>XYL-8XI-ΔISU1>XYL-8XI-ΔISU1>xyl-WT. These results indicated that
NFS1
gene mutation significantly improved xylose metabolic capacity, while ISU1 gene knockout had a specific
effect
on xylose metabolism, but it was not as significant as NFS1 mutation. Through this experiment, we have
identified promising targets for xylose metabolism enhancement.

![](https://static.igem.wiki/teams/5351/result20.png)

Figure 20. The xylose plate detected xylose metabolism.

###### b) Using HPLC to analyze the fermentation of strains

High-performance liquid chromatography (HPLC) testing will further quantitatively analyze the xylose
metabolism to verify the actual performance of different strains in xylose metabolism. The strains were
fermented in a YPDX medium to evaluate their actual xylose metabolism efficiency. The results of HPLC will
further support the advantages of NFS1 mutant strains in xylose metabolism, and these results provide new
ideas and a theoretical basis for yeast strains to optimize xylose utilization.

The concentrations of xylose were measured using an HPLC system (Waters e2695) equipped with
an Aminex HPX-87H
ion exchange column (300 × 7.8 mm; Bio-Rad) at 35°C and a Waters 2414 refractive index detector. Sulfuric
acid
(5 mM) served as the mobile phase with a flow rate of 0.6 ml min−1 [6]. The biological company conducted the
specific testing.

From Figure. 21, it is evident that introducing xylose isomerase into different parent strains resulted in
enhanced xylose metabolism capabilities. Further validation of the NFS1 mutant strains' advantages in xylose
metabolism was achieved through the actual xylose metabolism performance in the fermentation broth. The
strain
with the fastest xylose utilization rate nearly depleted the xylose in the medium within 40 hours.
Additionally, observations on strain growth revealed that the modified strains exhibited minimal impact on
growth, with an earlier onset of exponential growth. This rapid growth of the strains also positively
influenced xylose utilization.

![](https://static.igem.wiki/teams/5351/result21.png)

Figure 21. Comparison of xylose metabolism and growth status of the modified strains (a: Xylose metabolism
profiles of different strains; b: Growth status comparison among different strains)

The ultimate goal of enhancing xylose utilization in our engineered strains is for ethanol production.
Therefore, we also measured the ethanol content in the fermentation broth after fermentation to assess the
potential of our strains in the field of second-generation biofuel ethanol production. Experimental results,
as shown in Table 1, indicate a significant improvement in ethanol production capacity in the engineered
strains. Notably, our strain xyl-8XI-nfs1 exhibited a 5.2-fold increase in ethanol production compared to
the
parental strain, reaching 662.18 mg/L. This result further validates the success of our genetic
modifications
to the strains.

###### Table 1. The ethanol production levels of different strains (48 h)

|  |  |
| --- | --- |
| Strains | Ethanol concentration (mg/L) |
| xyl | 106.72 |
| xyl-8XI | 298.74 |
| xyl-8XI-nfs1 | 662.18 |
| xyl-8XI-△ISU1 | 367.16 |
| xyl-8XI-nfs1-△ISU1 | 307.10 |

Discussion

In our experiment, we utilized YPDX medium rather than glucose and xylose derived from lignocellulosic
biomass hydrolysate. Therefore, in the future, we will need to ferment with actual hydrolysate, which
contains not only glucose and xylose but also a significant amount of inhibitors generated from lignin
decomposition. This poses specific requirements for the stress tolerance of our strains [7,8]. We aim not
only to enhance the strains' xylose utilization capability but also to increase their robustness in the
hydrolysate, ultimately facilitating improved production of second-generation biofuel ethanol.

References

[1] Li X, Wang Y, Li G, et al. Metabolic network remodelling enhances yeast's fitness on
xylose using aerobic
glycolysis[J]. Nature Catalysis, 2021, 4(9): 1-14.

[2] Zhu L, Li P, Sun T, et al. Overexpression of SFA1 in engineered Saccharomyces cerevisiae
to increase
xylose utilization and ethanol production from different lignocellulose hydrolysates[J]. Bioresour Technol,
2020, 313: 123724.

[3] Shaobo Liang, Yinglei Han, Liqing Wei, et al. Production and characterization of bio-oil
and bio-char from
pyrolysis of potato peel wastes[J]. Biomass Conversion and Biorefinery, 2015, 5(3): 237-246.

[4] Matuštík Jan, Kočí Vladimír. Does renewable mean good for the climate? Biogenic carbon
in climate impact
assessments of biomass utilization[J]. GCB Bioenergy, 2022, 14(4): 438-446.

[5] Meher L C, Sagar D V, Naik S N. Technical aspects of biodiesel production by
transesterification—a
review[J]. Renewable and Sustainable Energy Reviews, 2006, 10(3): 248-268.

[6] Wei Fangqing, Li Menglei, Wang Ming, et al. A C6/C5 co‐fermenting Saccharomyces
cerevisiae strain with the
alleviation of antagonism between xylose utilization and robustness[J]. GCB Bioenergy, 2020, 13(1): 83–97.

[7] Brat D, Boles E, Wiedemann B. Functional expression of a bacterial xylose isomerase in
Saccharomyces
cerevisiae[J]. Applied Microbiology and Biotechnology, 2009, 75: 2304-2311.

[8] Liu CG, Xiao Y, Xia XX, et al. Cellulosic ethanol production: Progress, challenges and
strategies for
solutions[J]. Biotechnol Adv, 2019, 37(3): 491-504.

{% endblock %}