iGEM 2022 — Team nefu-china

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

##

Here is an overview over the major experiments and accomplishments we had to manage during our project. We document the experimental processes and protocols in this Notebook.

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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# Engineering

Introduction

Cycle 1

Design

Build

Test

Learn

Cycle 2

Design

Build

Test

Learn

Cycle 3

Design

Build

Test

Learn

Reference

## Introduction

Welcome to our engineering page, where we present the engineering success of our case. The core theory of our case is derived from the YeastFab, a yeast DNA assembly strategy developed by Tsinghua University. Overall, based on the characteristics of our project, the engineering part is presented through multiple cycles. In each cycle, we will present our experimental design, construction process and validation assays according to the four basic processes of synthetic biology: Design-Build-Test-Learn.

In addition, the ultimate goal of our case is to significantly expand the technical concept of the YeastFab, achieve cross-species applications of the strategy, and significantly extend it to industrial production. To this end, we have made many attempts and efforts. We hope that any visitor on this page will be able to understand our engineering design, and every fan of synthetic biology will be inspired.

## Cycle 1

### Design

Up to now, we have found it very time-consuming and costly to construct a large number of DNA synthesis pathways simultaneously in real production. Although automated technology has given us a glimpse of the solution, machines are ultimately not humans and suffer from the lack of rational design thinking and the ability to handle emergencies. For this reason, we took note of the YeastFab technology developed by Tsinghua University, which can efficiently achieve large-scale DNA assembly. To accomplish efficient construction, we did the following design:

1. Based on the YeastFab technology, we replaced the mCherry red fluorescence gene by the ccdB toxin that can inhibit DNA unwinding activity as a new selection marker, which intuitively improved the efficiency of our screening and increased the rate to get positive clones.

2. We selected the integration site on the yeast genome, and homologous arms were designed at both ends of this site to guide targeted integration in the yeast genome.

3. To achieve high efficiency in DNA assembly using the YeastFab technology, we standardized our components by editing customized cleavage sites for BsaI and BsmBI to obtain customized sticky ends.

In our project, we demonstrated the process of our DNA assembly through the synthesis of lycopene.

### Build

Based on the YeastFab technology principle, we designed the pccdK2 series of vectors (pccdK2-P, pccdK2-O and pccdK2-T) carrying the basic standard parts and the TU series of vectors (TU1-11) with integrated transcription units.

We used different promoters to drive the expression of the three genes TmcrtE, BtcrtI, PacrtB in the lycopene synthesis pathway. A combination of a moderately strong promoter to induce crtE, a strong promoter to drive crtI, and a weak promoter to induce crtB achieved a relatively decent lycopene yield [1]. Therefore, we constructed the three promoters of different strengths, crtE, crtI and crtB, and the tCPS1 terminator in the pccdK2 family of vectors by the Golden Gate assembly method according to this collocation, i.e., pccdK2-pTEF1, pccdK2-pACT1, pccdK2-pFBA1; pccdK2-crtE, pccdK2-crtB, pccdK2-crtI; pccdK2-tCPS1. We designated these constructions as the Level 1.

Next, we built the Level 2 constructs based on the foundation of the Level 1. We used the TU family of vectors to assemble the components of the Level 1 plasmids by the Golden Gate assembly method to form the transcription units TU2-pTEF1-crtE-tCPS1, TU4-pACT1-crtB-tCPS1 and TU5-pFBA1-crtB-tCPS1.

We then assembled the transcriptional unit fragments from recombinant plasmids in the Level 2 (i.e., the cascade of genes in TU-P-crt-T described above) to form a metabolic pathway carrying the deficiency screening marker Ura, and added homologous arms at both ends of the pathway, which would guide this pathway to the desired locus in the yeast genome for homologous recombination. Experimentally, we transformed this metabolic pathway plasmid carrying the screening marker and homologous arms into yeast to allow it integrated into the genome.

![](https://static.igem.wiki/teams/4121/wiki/engineering/fig4.png)

**Figure 1.** The process of standardized DNA assembly strategy

### Test

We examined our integration results by three means.

1. We directly observed the integrated SD-Ura (Synthetic Dropout Media), and found a large amount of white colonies on the plates without any red one. The presence of colonies that grew normally in the deficient medium indicated that the screening marker gene was successfully integrated; however, lack of red colonies suggested that the synthetic pathway was either not integrated or not properly expressed.

2. We performed lycopene extraction from the colonies, and used HPLC to determine the presence of lycopene, which showed absence of detection.

3. We extracted the recombinant yeast genome and performed PCR to identify the integrated genes, and failed in identifying any inserted gene in the extracted yeast genomic DNA.

### Learn

As we analyzed the results of this round of engineering, we made a lot of conjectures: we doubted the accuracy or efficiency of the genomic PCR, metabolic overload that could possibly lead to mutations or loss of genes, impurity of the chassis cells, etc., but we could neither find any essence of the problem nor make any meaningful progress. It was not until we attended the CCiC conference (Conference of China iGEMer Community) in August 2022 that we received valuable advice from the jury: due to the active homologous recombination in Saccharomyces cerevisiae, it is highly possible that multiple genes will be lost if the same combination of terminators is repetitively used in an integrated metabolic pathway. Therefore, we decided to increase the specificity of the terminators in homologous recombination in order to avoid gene loss mediated by potential recombination caused by identical terminator sequences. Inspired by the Cycle 1, we changed the engineering protocol and started a second cycle.

## Cycle 2

### Design

In the second round of construction, we designed to make crtE, crtI and crtB link to three different terminators tPRM9, tCPS1 and tSPG5, respectively. In this way, we could prevent the loss of any integrated region in the yeast genome caused by homologous recombination in the subsequent synthesis pathway. Following the same construction strategy as the Cycle1, the recombinant plasmid pccdK2-tPRM9/tCPS1/tSPG5 was created by combining pccdK2-T as a vector with different terminators as complementary parts of the Level 1.

### Build

After completing the construction of the complementary parts as in Cycle 1 at the Level 1, we used this foundation to construct the following Level 2 in the Cycle 1, and generated TU2-pTEF1-crtE-tPRM9, TU4-pACT1-crtB- tCPS1 and TU5-pFBA1-crtB-tSPG5. Then, the target fragments in the Level 2 recombinant plasmids were assembled again to complete the Level 3 construction. The strategy was the same as that presented in Cycle 1.

![](https://static.igem.wiki/teams/4121/wiki/engineering/fig1.png)

**Figure 2.** Construction of transcription units after terminator replacements in level 2

### Test

We used the same methods as above to test the parts of this cycle:

1. Direct observation. At this time, we found a large amount of red colonies and a few white colonies by direct observation of the SD-Ura, which supported the prediction that homologous recombination was responsible for the unsuccessful integration observed in the Cycle 1.

2. We extracted the lycopene from the red colonies and measured the yield using HPLC, which indeed displayed the presence of this product.

3. We extracted the genome of the yeast cultivated from the red colonies. Using it as a template in PCR amplification, we detected predicted bands for the gene fragments that we attempted to integrate.

These results demonstrated that our second round of integration was successful and the integrated pathway could be successfully expressed in yeast.

### Learn

Although our integration was successful, we were not satisfied with the yield of lycopene, which did not meet our needs. After reflection and discussion, we realized that the cycle could be further optimized in two ways.

1. We could optimize the MVA synthesis pathway in the lycopene synthesis pathway.

2. We could explore the optimal incubation conditions of yeast.

These let us improve the protocol and start the third cycle (Cycle 3).

## Cycle 3

### Design

To further improve lycopene production, we interrogated the possibility of increasing its two precursors, IPP and DMAPP. For this purpose, we introduced the genes of tHMG and idi, two key enzymes in the MVA (mevalonic acid) pathway, into the original synthesis pathway. They could increase the consumption of Acetyal-CoA in the MVA pathway, and subsequently improve the productions of IPP and DMAPP, leading to enhanced lycopene production [2]. This strategy could enhance the synthesis of lycopene’s precursors, and theoretically increase the yield of lycopene.

### Build

In the Cycle 3, we constructed pccdK2-tHMG and pccdK2-idi at the Level 1, TU2-pACT1-crtB-tCPS1, TU4-PA-tHMG-tTDH1 and TU5-PB-IDI-tADH1 at the Level 2, carrying a Leu screening marker using the same method as the two cycles above. We continued to ligate parts through the Golden Gate, using the transcription unit where crtB is located as the upstream homologous arm and the homologous arm of the original pathway as the downstream homologous arm for integration, allowing for repetitive recombinant integration at the same genomic locus. In this process, a selection marker was replaced, allowing us to make theoretically "countless" additions, knockouts or substitutions of transcription units with a limited number of selection markers.

![](https://static.igem.wiki/teams/4121/wiki/engineering/fig2.jpg)

**Figure 3.** Schematic representation of the yeast genome superposition principle.

### Test

We extracted the genomic DNA of the yeast cultivated from the red colony for PCR. However, the PCR amplification always showed poor yields, which would need to be optimized in the following studies. On the other hand, the integrated fragments of crtE and idi genes from the first and second round of integrations were much easier to be amplified by PCR, indicating the success of our two rounds of integrations. We also extracted lycopene from the red colonies and measured the yield using HPLC. The yield of lycopene increased substantially from 0.56 mg/g DCW (dry cell weight) to 1.52 mg/g DCW, with an approximate 2.7-fold increase. These results demonstrated that the lycopene synthesis pathway was successfully optimized and the yield was improved through the two-round integrations.

### Learn

After the two rounds of integrations described above, we found that the yield of lycopene was increased substantially. However, based on the experience of the second round of integration, we can continue the integration operation in the future and add other key enzyme genes in the synthesis pathway in addition to tHMG and IDI. This may help us to obtain optimal pairings to achieve the highest yield, with moderately increased number of integrated genes. It is also possible to optimize the lipid metabolism process by introducing the sterol ester synthesis genes ARE1 and ARE2, thus promoting lycopene production [3].

## References

1. Bahieldin, A., Gadalla, N. O., Al-Garni, S. M., Almehdar, H., Noor, S., Hassan, S. M., Shokry, A. M., Sabir, J. S., & Murata, N. (2014). Efficient production of lycopene in Saccharomyces cerevisiae by expression of synthetic crt genes from a plasmid harboring the ADH2 promoter. Plasmid, 72, 18–28. https://doi.org/10.1016/j.plasmid.2014.03.001
2. Cheng, T., Wang, L., Sun, C., & Xie, C. (2022). Optimizing the downstream MVA pathway using a combination optimization strategy to increase lycopene yield in Escherichia coli. Microbial cell factories, 21(1), 121. https://doi.org/10.1186/s12934-022-01843-z
3. Zhao, Y., Zhang, Y., Nielsen, J., & Liu, Z. (2021). Production of β-carotene in Saccharomyces cerevisiae through altering yeast lipid metabolism. Biotechnology and bioengineering, 118(5), 2043–2052. https://doi.org/10.1002/bit.27717

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# Results

Level 1

Level 2

Level 3

Cross-species

References

## Level 1

# Promoter

### Design of Promoter Screening and Characterization of Their Strengths

First of all, we downloaded the sequences of several promoters in the yeast genome from the EPD
(Eucaryotic promoter database). After excluding the promoters with the cutting sites of the restriction
enzyme BsmBI and BsaI, we identified 29 promoters. In order to clone each promoter into the target
vector, we used a pair of primers with specific prefix sequences to individually amplify the promoters.
After obtaining the promoter fragments, the sequences of the ccdB in the vector of TU11-ccdB-mcherry-T4
were replaced by the sequences of target promoters, and thus the TU11-promoter-mcherry-T4 plasmids were
constructed through the Goldengate method, followed by transformation into *S. cerevisiae* (Figure 1). The
strength of each promotor was characterized by their ability in enhancing the fluorescence intensity of
mCherry (Figure 2).

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-1.png)

**Figure 1.**  Yeast characterization plates. Images of Petri dishes with yeast
colonies: the difference of mCherry fluorescence intensity can be clearly visualized when exposed to
blue light.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-2.jpg)

**Figure 2.**  Strengths of promoters. The data of mCherry fluorescence intensity were
obtained from the reporters using the different promoters.

### Construction of Standardized Promoters

After finishing strength characterization of 29 promoters, we selected 10 of them with relatively high
strengths, and integrated them into the pccdK2-P vector by the Goldengate method to construct the
pccdk2-promoter. For each primer, the nucleotides (nts) in green text indicate the BsaI recognition
sequences, while the nts in red text represent the sticky ends after the digestion of BsaI. The bases with fluorescent yellow are extra sequences to increase the efficiency of digestion, and “nnnn…nnn” represents specific sequences to amply your target.
For different targets, only the
"nnnn...nnn" region needs to be changed. We used the following primers to amplify the promoters.

5':**agcgtg**
GGTCTCgGGCTnnnnnnnnnnnnnnnnnnnn

3':**gtgctg**
GGTCTCaCATCnnnnnnnnnnnnnnnnnnnnn

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-3.png)

**Figure 3.** Agarose gel electrophoresis images for the PCR products of the
standardized parts. From The constructions are for the pccdK2-P1 (left) and pccdK2-P5 (right). The
predicted size of the PCR band is 536 bps. The average positive rate was over 90%. NC: negative
control without any template.

### Optimization of the PGK1 Promoter Truncation

We noticed that the PGK1 promoter in the part library of the iGEM official website had a length of
1,500 bps, which could be relatively too long when used as a transcription unit. Therefore, we conducted
experiments of PGK1 promoter truncation. We amplified the PGK1 promoter sequence from the yeast genome
with different lengths of 200, 400, 600 and 800 bps, and determined their relative strengths (Figure 4).

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-4.jpg)

**Figure 4.** Characterization of the strengths of the truncated PGK promoters.

# ORF

### Construction of Standardized ORF

In order to prove the efficiency of the YeastFab assembly strategy and integrate the lycopene
production system into the genome of *S. cerevisiae*, we first identified the key enzyme genes crtB, crtI
and crtE in the lycopene synthesis pathway, and then added the genes of terpenoid synthesis precursors
in the MVA pathway to increase the production. We also used the Goldengate approach with the restriction
enzyme BsaI to insert the target gene into pccdK2-O. The primers used to amplify the target gene is
shown as follows. We have built a total of 10 standard plasmids of ORFs. The glue diagrams of colony PCR
are correct (Figure 5）.

5':**agcgtg**
GGTCTCaGATGnnnnnnnnnnnnnnnnnnnn

3':**gtgctg**
GGTCTCgGCTAnnnnnnnnnnnnnnnnnnnnn

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-5.png)

**Figure 5.**  Colony PCR to screen for the positive clones in the construction of
standard parts of the lycopene synthesis pathway. The predicted PCR band sizes are: (a) pccdK2-tHMG:
1,715 bps; (b) pccdK2-crtE: 1,310 bps; (c) pccdK2-ERG10: 1,328 bps; (d) pccdK2-ERG19: 1,319 bps. NC:
negative control without any template.

# Terminator

### Construction of Standard Parts of Terminators

In this part, we used the same method as that in the promoter construction. We selected 10 terminators,
and used the following primers to amplify the terminators.

5':**agcgtg**
GGTCTCtTAGCnnnnnnnnnnnnnnnnnnnn

3':**gtgctg**
GGTCTCgGAGGnnnnnnnnnnnnnnnnnnnn

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-6.png)

**Figure 6.** The result of colony PCR in constructing the standard plasmids of
terminators. 1-5 are for the TADH1 colony PCR with a predicted band of 356 bps; 6-10 are for the TENO1
colony PCR with a predicted band of 356 bps. NC: negative control without any template.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/1.png)

**Figure 7.** Positive rates of the Level 1 constructions. Different colors refer to the
percentages of colony PCR positive rates achieved during the construction of the Level 1 standard
components. For example, red refers to the percentage of the number of constructions with 100%
bacterial P positive rate in the total number of constructions.

## Level 2

# Constructions of Transcription Unit

It was the time to assemble the transcription units when all the desired parts were successfully
constructed. In this level, the promoter, ORF and terminator of interests were released from their
vectors, and assembled together into an accepting vector in a one-pot reaction[1]. In the first round of transcription unit
construction, we selected 5 promoters of different strengths (pccdk2-P1, P5, P7, P8 and P20), and the
same terminator pccdk2-T4 to combine with pccdk2-crtE, crtI and crtB for assembly using BsmBI digestion.
The colony PCR agarose gel electrophoresis for the screening of the transcription unit is shown below.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-8.png)

**Figure 8.** Representative agarose gel electrophoresis images of the colony PCR
products in the construction of TU vectors. The predicted sizes of the PCR products are: (a)
TU2-P1-crtE-T4: 2,104 bps; (b)TU5-P1-crtB-T4: 1,813 bps; (c) TU2-P7-crtE-T4: 2,105 bps;
(d)TU2-P5-crtE-T4: 2,105 bps. NC: negative control without any template.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-9.png)

**Figure 9.**  The Positive rates of the Level 2 constructions. It can be seen that only
a small number of constructions in the Level 2 had positive rates below 80%, and the overall
constructions at this level were very efficient.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-10.png)

**Figure 10.** . Image of agarose gel electrophoresis of
plasmid digestion to verify inserted DNA fragments. The blue lines denote the bands with predicted
sizes of 4,964 bps and 2,010 bps(1-2 for TU2-P1-crtE-T4, 3-4 for TU2-P5-crtE-T4, 5-6 for
TU2-P7-crtE-T4, 7-8 for TU2-P8-crtE-T4, 9-10 for TU2-P20-crtE-T4). The orange lines denote the bands
with predicted sizes of 4,964 bps and 2,577 bps (11 for TU4-P1-crtI-T4, 12 for TU4-P5-crtI-T4, 13 for
TU4-P7-crtI-T4, and 14 for TU4-P8-crtI-T4). The last pink lines denote the bands with predicted sizes
of 4,964 bps and 1,719 bps. (15-16 for TU5-P1-crtB-T4, 17-18 for TU5-P5-crtB-T4, 19-20 for
TU5-P7-crtB-T4, 21-22 for TU5-P8-crtB-T4, and 23 for TU5-P20-crtB-T4). NC: negative control without
any template.

## Level 3

# Integration of the Lycopene Synthesis Pathway

To integrate the lycopene synthesis pathway into the yeast genome, we chose to transform gene clusters
carrying selection markers and homology arms into yeast. As the yeast used by us was a kind of
four-deficient strain lacking the ability to synthesize three different amino acids and uracil, we
cultivated the yeast in a nutrient-deficient medium, and used the synthetase genes corresponding to
these four substances as selection markers to screen for yeast colonies with successful integration of
the lycopene gene cluster (Figure 11).

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-11.jpg)

**Figure 11.**  Schematic representation of homology arm integration of the lycopene
gene cluster into the genome of *S. cerevisiae*.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-12.png)

**Figure 12.**  Images of Petri dishes of the yeast with the integration of the lycopene
synthesis pathway using the homology arms. (a) The experiments using the OGG homology arm; (b) The
experiments using the APL4 homology arm; (c) The experiments using the TY homology arm with
multi-integration sites. The yeast colonies of both OGG and APL4 were white, only the TY homologous
arm integrated appeared as red colonies.

Based on Figure 12, all yeast colonies generated by the homologous arms with a single integration site
were white, while the experiments using the TY homologous arms with multi-integration sites showed red
colonies. It was obvious that our integration did not succeed according to the experimental results.
Around this time, we participated in the CCIC at that time. The judges of the conference suggested us to
use distinct terminators in different transcription units, because the use of the same terminator in
different units may cause gene loss due to the strong recombination ability of *S. cerevisiae*. We
accepted this valuable suggestion, and planned to use different terminators. We hoped that the new
strategy could solve the problems of yeast redness and transmission degradation.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-13.png)

**Figure 13.**  Schematic representation of gene loss when an identical terminator is
used in two transcription units after integrated into the yeast genome. Homologous recombination may
occur when both transcription units use T4 as their terminators, leading to the loss of the crtI gene.

After identifying the reason for the unsuccessful integration of the lycopene synthesis pathway into
the yeast genome, we immediately reconstructed a series of transcription units with different
terminators, and integrated them into the yeast genome through a single integration homologous arms.
Ultimately, we obtained a petri dish with a large number of red colonies. We picked the red colonies and
expanded them in YPD. We extracted the genomic DNA to verify the successful integration of the lycopene
synthesis pathway. Meanwhile, we also determined the yield of lycopene by HPLC analysis.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-14.png)

**Figure 14.**  Schematic diagram of the integration of the lycopene synthesis pathway
in yeast. The genes marked with blue borders below are some of the genes that we designed primers to
verify by PCR amplification.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-15.png)

**Figure 15.**  URR-crtEIB. Image of a Petri dish containing red colonies after
different
terminators were used to integrate the lycopene synthesis pathway.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-16.png)

**Figure 16.** Agarose gel analysis of PCR amplification on extracted yeast genomic DNA to verify
the integration of the lycopene synthesis pathway. Agarose gel electrophoresis of colony PCR extracted
genome, 1-4 corresponding to the gene fragments marked in blue borders in Figure 14 respectively.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-17.png)

**Figure 17.**  Standard curve of lycopene. Lycopene has a maximum absorption peak at
the wavelength of 474 nm. The absorption peak area of different concentrations of lycopene standards
at 475 nm was measured, and the standard curve of the proportional relationship between the peak area
and the concentration was obtained.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-18.png)

**Figure 18.**  Elution process of HPLC to detect the production of lycopene. The figure
above shows the lycopene yield of the strain of URR-crtEIB.

**Table 1.** Lycopene production by the corresponding strains.

| Sample name | Output |
| --- | --- |
| URR-crtEIB | 1.5 mg/g DCW |

# Response Surface Method

In the process of testing the integrated lycopene synthesis pathway, we found that the output did not
meet up with our expectations. Therefore, we attempted to improve the yield from the perspective of
fermentation. We modeled the fermentation process to find optimized conditions. We reviewed the
literature and discovered that optimization through Response Surface Methods (RSM) was a common method.

**Table 2.** Experimental protocols for the three factors in the
fermentation.

![](https://static.igem.wiki/teams/4121/wiki/model/model1.png)

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-19.png)

Y=5.5+0.2\*A+0.15\*B+0.25\*C+0.01\*A\*B-0.14\*A\*C-0.01\*B\*C-0.05\*A^2-0.0008\*B^2+0.028\*C^2-0.0008\*A^2\*B+0.0223\*A^2\*C-0.000042\*A\*B^2

**Figure 19.**  (a) 3D plot of the effects of glucose concentration and dissolved oxygen
on lycopene yield; (b) 3D plot of the effects of dissolved oxygen and inoculum on lycopene yield; (c)
3D plot of the effects of glucose concentration and inoculum on lycopene yield.

# Superimposed Yeast Genome

The yield of lycopene by the engineered yeast was about 1.5 mg/g DCW as quantified by HPLC, which did
not
meet our satisfaction. Therefore, we planned to optimize the upstream of the MVA pathway. In addition to
integrating the crtE, crtI and crtB into the yeast genome, we carried out the second round of
integration of the genes of tHMG and IDI. For this purpose, we used the crtB as the upstream homologous
arm, and a previous homologous arm as the downstream homologous arm. It was interesting to employ such a
reorganization method, which allowed us to use limited filter tags to superimpose and integrate
infinitely by different DNA fragments.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-20.jpg)

**Figure 20.**  Schematic representation of the yeast genome superposition principle.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-21.png)

**Figure 21.**  Images of cultured yeast cells derived from the red colonies of the
yeast with the superposed genome.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-22.png)

**Figure 22.**  Chromatographic elution of HPLC to determine the lycopene yield in the
yeast cells derived from red colonies on transformed petri dishes of the yeast with superimposed
genome. The figure above shows the lycopene yield of the strain of URR-crtEIB-tHMG-IDI.

**Table 3.** Production of lycopene by the URR-crtEIB-tHMG-IDI.

| Sample name | Output |
| --- | --- |
| URR-crtEIB-tHMG-IDI | 3.6 mg/g DCW |

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-23.png)

**Figure 23.** A dedication map of Northeast Forestry University (NEFU) to the
70th
anniversary using the lycopene product by engineered yeast strains. We would like to pay tribute to
the forerunners who made outstanding contributions to the establishment and development of Northeast
Forestry University, and also send a congratulation gift to our team of the NEFU\_China for the
th
anniversary of NEFU.

# Explore the Integration Efficiency of Plasmid and Homologous Arms：

In order to explore the advantages and disadvantages of homologous arm
integration and plasmid
integration, we used the URR[2]
homologous arms that are located in open chromatin area, or a series of TU plasmids to integrate
mCherry into WAT21. As a result, we obtained correctly integrated strains by screening nutritionally
deficiency markers and examining the fluorescence intensity of mCherry.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-24.png)

**Figure 24.**  (a) Growth of URR1-P1-mCherry-T1-Ura3-URR2 integrated yeast after 96 h
of cultivation. (b) Growth of TU-P1-mCherry-T1 integrated yeast after 96 h of cultivation. (c)
Yellow：Characterization of URR1-P1-mCherry-T1-Ura3-URR2 mCherry. Green：Characterization of
TU-P1-mCherry-T1 mCherry.

Based on the graphs of Figure 24 for the primary integration strains, the
plasmid-integrated strains had excellent integration efficiency and decent mCherry expression capacity,
compared to the URR-integrated strains.

Due to the low phenotypic correctness of the URR integrated strains, we needed
to further confirm whether all URR integrated strains had been correctly integrated. We then selected
two red and two white colonies to extract their genomic DNA, employed PCR to amplify both ends of the
homology arm, and then use the PCR products to further confirm the presence of the mCherry gene by
additional PCR amplification.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-25.png)

**Figure 25.**  Agarose gel electrophoresis of the PCR products to determine the
presence of the URR homology arm and the mCherry gene; (a) 1-2 are red colony genomes, 3-4 are white
colony genomes. PCR was performed at both ends of the URR homology arm, and the correct band was
2800bps; (b) The product of PCR at both ends of the URR homology arm was recovered and PCR was
performed with primers at both ends of the mCherry gene, and the correct band was 750bps.

In the PCR amplification to determine the presence of the URR integrated
mCherry, the yeast cells from the red and white colonies showed the same bands with the predicted sizes,
suggesting that the white clones underwent correct integration but did not have the desired phenotype.
To investigate the genetic stability of the plasmid-integrated strain and the homologous arm-integrated
strain, we cultured and characterized the URR-integrated strain 3 and the plasmid-integrated strain 2 by
passaging.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-26.png)

**Figure 26.**  Schematic representation of plasmid integration and URR integration of
the mCherry gene in subcultured yeast.

MCherry expression in the plasmid integrated yeast gradually decreased after
subculture, and its level in the fifth passage was less than 40% of the first generation, while mCherry
expression in the URR integrated yeast increased steadily after subculture. When the characterized
mCherry expression of the primary generation was low, the level of the fifth generation could be
improved by over 2 times. These results indicated that the integration of exogenous genes into the yeast
genome by the homology arms had good genetic stability.

# Comparison of Different Methods of mCherry Gene Integration.

We used four methods to characterize the mCherry gene integration in the yeast
genome and its fluorescence levels to compare the integration efficiency and characteristics of
different integration methods.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-27.png)

**Figure 27.**  Growth of transformed yeast in 96h culture: (a) HapAmp-P1-mCherry-T1 (b)
URR1-P1-mCherry-T1-Ura3-URR2 (c) His-1-P1-mCherry-T1-Ura3-His-2 (d) TU-P1-mCherry-T1.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-28.png)

**Figure 28.**  Comparison of mCherry fluorescence intensity of 4 different yeast clones
with mCherry integrations using 4 different methods.

The comparison of the characterization with the four integration methods
showed that the integration efficiency of the integration site in open chromatin area was better than
that using common homologous arm, while the integration efficiency and expression capacity of the
self-replication site were improved compared to those using other integration sites. The results
indicated that different integration sites could lead to distinct integration and expression
efficiencies. However, we can choose to integrate a target gene into different sites of the genome to
improve its integration and expression efficiency.

To determine whether the HapAmp locus is integrated and self-replicating, we
extracted the genomic DNA, and conducted identification PCR at both the arm1 and arm3 ends.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-29.png)

**Figure 29.**  Agarose gel electrophoresis for the PCR products to test different
integration. Lane 1 was that from WAT21 original strain, while lanes 2 and 3 were the
genomes extracted from HapAmp-P1-mCherry-T1 integrated strains. The PCR band without integration was
predicted to be 2,315 bps, while lanes 2 and 3 should have predicted bands of around 9,000 bps,
indicating that the target gene was successfully integrated into the target site, and was replicated
at least once.

## Cross-species Application

We hope to extend the YeastFab technology to cross-species applications. We
still performed the Level 1 and Level 2 constructions sequentially in the universal system, assembled
the plasmid carrying the metabolic pathway in vitro, and conducted a double plasmid transformation with
the plasmid carrying the Cas9 gene, which cleaves the genome to obtain a cut where the metabolic pathway
could be integrated. This integration strategy is slightly different from that of *S. cerevisiae*, but we
could still achieve efficient assembly of the target metabolic pathway using the YeastFab strategy. The
successful integration was verified by TLC assays and the production of the relevant metabolites,
indicating that this synthetic pathway was also successfully expressed in *Aspergillus oryzae*. Therefore,
we have achieved the cross-species application of the YeastFab technology.

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-30.png)

**Figure 30.**  Agarose gel image of restriction enzyme digested plasmids. Line
1/2：Digestion of SalI: predicted bands are 316, 8494 and 6316 bps. Line 3/4：Digestion of HindIII:
predicted bands are 11541 and 3583 bps. The plasmids assembled in vitro were digested, and the
positive rate was 100%

![](https://static.igem.wiki/teams/4121/wiki/zqh-results/results-31.png)

**Figure 31.** Results of TCL detection. In each of the 7 groups, the left well is the negative control sample, and
the right well is the experimental sample. Only one experimental group (the third from the left)
produced an irrelevant product. The integration efficiency of the gene clusters was 94.117%

## References

1. Jiang, S., Luo, Z., Dai, J. (2021). Use YeastFab to Construct Genetic Parts and
   Multicomponent Pathways for Metabolic Engineering. In: Xiao, W. (eds) Yeast Protocols. Methods in
   Molecular Biology, vol 2196. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0868-5\_13
2. Yakun Guo, Junkai Dong, Tong Zhou, Jamie Auxillos, Tianyi Li, Weimin Zhang, Lihui Wang, Yue
   Shen, Yisha Luo, Yijing Zheng, Jiwei Lin, Guo-Qiang Chen, Qingyu Wu, Yizhi Cai, Junbiao
   Dai.（2015）YeastFab: the design and construction of standard biological parts for metabolic engineering
   in *Saccharomyces cerevisiae*. Nucleic Acids Research, 43,88. https://doi.org/10.1093/nar/gkv464

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