iGEM 2023 — Team scu-bse-china

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{% block title %}Notebook{% endblock %}
{% block lead %}"The notebook wiki page is where our ideas take shape, our mistakes find resolution, and our journey unfolds in ink."{% endblock %}
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[notebook.pdf](https://static.igem.wiki/teams/4937/wiki/notebook/hp-notebook.pdf)

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}"Engineering is the art of turning imagination into innovation."{% endblock %}
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#### Engineering

- [1. Fusion PCR](#part1)
- [2. Yeast Transformation](#part2)
- [3. Ployamine production](#part3)
- [4. REFERENCE](#part4)

Here, we will demonstrate how DBTL (Design-Build-Test-Learn) principles play a pivotal role in guiding experiments.

**1. Fusion PCR**

The fusion of multiple DNA fragments is a commonly used technique in building yeast cell factories and is a core technology in synthetic biology. In our efforts to construct expression cassettes for genes from different sources, we employed the DBTL approach to discover an efficient method for conducting fusion PCR. To assemble the OAZ1 \_upstream-TEF1p-SPE1-PRM9t-Oaz1 downstream fragment, we initially obtained the individual subfragments through genomic PCR. Subsequently, we built these subfragments through fusion PCR.

First Round:

①PCR without primers:

| Component | Volume(μL) | Final concentration |
| --- | --- | --- |
| 2×KOD One PCR mix | 12.5 |  |
| OAZ1\_upstream(493bp,GC35%) | x | 15.6ng |
| TEF1p | y | 38.2ng |
| SPE1 | z | 228.7ng |
| PRM9t | a | 23.2ng |
| OAZ1\_downstream(494bp,GC36%) | b | 15.7ng |
| ddwater | 12.5-x-y-z-a-b |  |
| Total | 25 |  |

Process:

| Temperature(℃) | Time(s) |
| --- | --- |
| 98 | 10 |
| 58 | 5 |
| 68 | 20 |
|  | ×15 Cycle |

②PCR with primers:

| Component | Volume(μL) | Final concentration |
| --- | --- | --- |
| uph -fw | 0.75 | 0.3μM |
| downh-rv | 0.75 | 0.3μM |
| 2×KOD One PCR mix | 12.5 |  |
| Product in PCR without primers | 2 |  |
| ddwater | 9 |  |
| Total | 25 |  |

Process:

| Temperature(℃) | Time(s) |
| --- | --- |
| 98 | 10 |
| 50 | 5 |
| 68 | 20 |
|  | ×30 Cycle |

Build: We constructed as Design.

Test: The gel electrophoresis results in Figure 1 indicate that the fragment size is about 1000bp-2000bp, however, our target fragment is 3003bp, which means that it doesn’t align with our expectations.

![figure1.png](https://static.igem.wiki/teams/4937/wiki/engineering/p1.png)

Figure 1

Learn: We suspected that this discrepancy may be related to the low annealing temperature used in the second step of primer PCR, leading to non-specific amplification (as seen in the bands between 1000-2000bp in the gel). Therefore, in the next round of experiments, we plan to increase the annealing temperature.

Second Round:

* Design: We have adjusted the annealing temperature to 58℃ while keeping the other conditions unchanged.
* Build: We constructed as Design.
* Test: The gel electrophoresis results in Figure 2 indicate that the fragment size isn’t align with our expectations.
![figure1.png](https://static.igem.wiki/teams/4937/wiki/engineering/p2.png)

Figure 2

* Learn: We suspected that this issue may be related to the sensitivity of the polymerase to primer-template binding or the specificity of the primers. Therefore, we plan to test with an enzyme that is less sensitive to this binding.

Third Round:

* Design: We have switched to PrimeSTAR Max Premix (2X) for the PCR mix, and for both rounds of PCR, we have adjusted the annealing temperature to 55℃ for experiment.
* Build: We constructed as Design.
* Test: The gel electrophoresis results in Figure 3 indicate that the fragment size is align with our expectations.
![figure1.png](https://static.igem.wiki/teams/4937/wiki/engineering/p3.png)

Figure 3

* Learn: The efforts outlined above have enabled us to develop an efficient method for multi-fragment fusion. We have summarized and compiled this method into a comprehensive operating manual, which can be found on [Contribution](https://2023.igem.wiki/scu-bse-china/contribution) for reference and dissemination.

**2. Yeast Transformation**

Yeast transformation is the first crucial step in yeast engineering applications, and achieving high transformation efficiency facilitates rapid experimentation. To construct various yeast strains, we made several attempts to optimize the yeast transformation method. Our strains are uracil-deficient, and our plasmids contain URA3, which can complement this deficiency. Therefore, only successfully transformed strains can grow on Delft solid plates and be expanded in Delft liquid culture medium.

First Round:

* Design: For our initial yeast transformation attempts, we followed the instructions provided in the manual. When plating on the final plates, we diluted the yeast transformation mixture 50-fold.
* Build: We constructed as Design.
* Test: After 48 hours, numerous small colonies grew on the plates (Figure 4A). We picked clones and cultured them in liquid medium with the same composition, and after another 48 hours, there were no signs of turbidity in the medium (Figure 4B).
![figure1.png](https://static.igem.wiki/teams/4937/wiki/engineering/p4.png)

Figure 4

* Learn: The fact that yeast on the plates after 48 hours was much smaller in number compared to the wild-type yeast and that liquid culture did not promote growth strongly suggests that these strains were likely false positives. However, the exact reasons for this were not easy to pinpoint. To validate whether this was related to low transformation efficiency and our excessive dilution, we made improvements targeting these two aspects.

Second Round:

* Design: We plan to plate the same volume of transformation mixture without dilution onto the plates.
* Build: We constructed as Design.
* Test: After 48 hours, there were very few larger clones along with numerous small clones that grew on the plates (Figure 5A). We picked clones of different sizes and cultured them in liquid medium with the same composition. After another 48 hours, only the medium from the large clones became turbid, while the medium from the small clones remained clear (Figure 5B).
![figure1.png](https://static.igem.wiki/teams/4937/wiki/engineering/p5.png)

Figure 5

* Learn: This indeed indicates that the generation of small clones was a result of false positives, and our previous dilution led to a decrease in positive rate. We speculated that the production of small clones may have occurred because we used Delft+Uracil medium when preparing yeast competent cells, leading to the accumulation of a substantial amount of uracil in some cells. These cells, after a few generations of division, formed visible clones. Therefore, through our two rounds of iteration, we have successfully found an efficient yeast transformation method.

**3. Ployamine production**

Our project aims to enhance the thermo-tolerance of *S. cerevisiae*, so we firstly plan to enhance the flux of polyamine synthesis in our strain. OAZ1 restricts the synthesis of polyamine, thereby limiting the direct precursor spermidine's production.

First Round:

* Design: We hypothesis the knockout of endogenous OAZ1 may open the flux to polyamines synthesis. So we design the gene editing experiment to decreased the effect of OAZ1 by CRISPR/Cas9 technology.
* Build: We built this strain using the method as literature reported1.
* Test: After constructing the strain, we performed HPLC test to examine the production of putrescine and spermidine(Figure 6).
![figure1.png](https://static.igem.wiki/teams/4937/wiki/engineering/p6.png)

Figure 6

* Learn: The putrescine and spermidine are increased from 1.30 mg/L and 2.90 mg/L to 5.70 mg/L and 7.15 mg/L after 48h cultivation, respectively. To further enhance the flux of polyamine, we decided to expand the flux of entrance of polyamine in the next round.

Second Round:

* Design: SPE1 is an ornithine decarboxylase, which catalyses the ornithine decarboxylation reaction that is the committed step for synthesis of putrescine in Saccharomyces cerevisiae. Thus, we design the gene editing experiment to make OAZ1Δ::SPE1 strain by CRISPR/Cas9 technology.
* Build: We built this strain using the method as literature reported[1](#ref1).
* Test: After constructing the strain, we performed HPLC test to examine the production of putrescine and spermidine(Figure 7).
![figure1.png](https://static.igem.wiki/teams/4937/wiki/engineering/p7.png)

Figure 7

* Learn: The putrescine and spermidine are increased to 9.5 mg/L and 15.2 mg/L further. Until this, we successfully engineered the strain to produced a considerable quantity of polyamines.

**4. REFERENCE**

1. Mans, R., van Rossum, H. M., Wijsman, M., Backx, A., Kuijpers, N. G., van den Broek, M., Daran-Lapujade, P., Pronk, J. T., van Maris, A. J., & Daran, J. M. (2015). CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS yeast research, 15(2), fov004. https://doi.org/10.1093/femsyr/fov004

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}"Experiments are the stepping stones to enlightenment."{% endblock %}
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#### Experiments

* [1. Fusion PCR](#part1)
* [2. HPLC for Polyamine Detection](#part2)
* [3. Quick Yeast DNA Extraction](#part3)
* [4. Yeast Transformation](#part4)
* [5. Delft Media Preparation](#part5)

**Here we show all standard protocols for our key experiments.**

**1. Fusion PCR**

Fusion PCR for less than 5 fragments:

1. Design the pathways by selecting the suitable promoters and terminators, and then design the primers
2. Cloning the genetic parts (Gene, promoters, terminators) by using the Prime-Star polymerase
   and corresponding Primers
3. One-pot fusion of the DNA fragments (promoter, gene and terminator). To ensure individual
   module functioning, each gene is linked with a promoter and a terminator. Moreover,
   homologous terminator of the front module or promoter of the next module was introduced at
   the 5’- or 3’-termini to mediate in vivo homologous recombination in the Pathway assembly step.
   Attention: The Phusion polymerase didn’t work well in the One-pot fusion amplification

   PCR procedure:

   1. Purified DNA fragments, promoter, functional gene, terminator and promoter of next module were mixed with a molar ratio at 1:3:5:7:XX:7:5:3:1, this mean the molar of DNA fragment increase from termini to middle with a arithmetic of 2. And the amount of terminal DNA amount is 50-100 ng/kb. This ensures the specific amplification.
   2. First round PCR without primers: Using this DNA mixture as a template and add to 12.5 µl 2×PrimerStar buffer, H2O to a total volume of 25 µl, then subjecte to PCR amplification with the thermocycle conditions of 95 ℃ for 3 min, 15 cycles of 98 ℃ for 10 s, 58 ℃ 15 s, 72 ℃ for 1 min/kb, at last 72 ℃ for 10 min.
   3. Second round PCR: Take 2 µl unpurified PCR products as template, add F- and R-primer and
      PrimeSTAR HS DNA polymerase and for normal PCR amplification in a total volume of 50 μl.
      Of course, the volume can be increased according to the needed DNA amount. Then subjected to PCR amplification with the thermocycle conditions of 95 ℃ for 3 min, 30 cycles of 98 ℃ for 10 s, 56 ℃ 15 s, 72 ℃ for 1 min/kb, at last 72℃ for 10 min.
   4. Verify and purify the PCR products with gel 2lectrophoresis
4. Pathway assembly. Equal molar amount of purified individual modules (100 ng/kb) and
   linearized plasmid (60-80 ng/kb) were mixed and transformed into S. cerevisiae with
   electroporation at 1.5 kV in a 0.2 cm gap electroporation cuvette using Bio-Rad Eporator.
5. Verification of the assembled pathways. Select colonies formed on the plates and culture in 5
   mL SC drop-out liquid medium at 30 ℃ for 72 h. Extract the plasmid and transformed into E. coli
   DH5α, recover the plasmid and verify the digest map using the specific restriction endonuclease.

**2. HPLC for Polyamine Detection**

0.1 mL samples from deep-well plates or fermenters were taken and subject to HW extraction (in this method, minimal medium was used instead of water to mitigate matrix effects). Tubes containing 0.9 mL of minimal medium were preheated in a heating block at 100℃ for 10 min. Then, the hot minimal medium was quickly poured over the 0.1 mL of liquid culture; the mixture was immediately vortexed, and the sample was placed in the 100℃ heating block for another 30 min. Subsequently, each tube was placed on ice for 5 min and then subject to centrifugation. The supernatants then stored in -20℃ or directly be subject to following quantification step. For extracellular polyamine detection, 1 mL samples from deep-well plates or fermenters were taken and subject to centrifugation. The supernatants then stored in -20℃ or directly subjected to the following quantification step.

Dansyl chloride derivatization based HPLC method was adopted for the detection of polyamines. Briefly, for derivatization, 0.125 mL of saturated NaHCO3 solution and 0.25 mL of dansyl chloride
solution (5 mg/mL in acetone) were added to 0.25 mL of sample (dilute samples whenever needed with minimal medium). Then the reaction mixture was incubated at 40℃ for 1 hour in the dark with occasional shaking. The reaction was stopped by adding 0.275 mLof methanol. Samples filtered through a 25 mm syringe filter (0.45 µm Nylon) can be used for HPLC detection. The following chromatographic condition are used: C18 (100 mm × 4. 6 mm; i. d., 2.6 μm; Phenomenex Kinetex), excitation wavelength 340 nm, emission wavelength 515 nm, sample injection volume 1.5 μL, column temperature 40℃, detector sensitivity was set at level 7, acquisition starts at 4.0 min. The mobile phase is water and methanol with the speed of 1 mL /min. The elution program is as follows: 50% to 65% methanol (0 to 5 min), 65% to 75% methanol (5 to 7.5 min), 75% to 87.5% methanol (7.5 to 9.5 min), 87.5% to 100% methanol (9.5 to 10.5 min), 100% methanol (10.5 to 11.5 min), 100% to 50% methanol (11.5 to 13.5 min), 50% methanol (13.5 to 16 min).

**3. Quick Yeast DNA Extraction**

1. Suspend single colonies (or 100 μL liquid YPD culture, OD600 = 0.4) in 100 μL 200 mM lithium acetate + 1% SDS solution;

2. Vortex and incubate for ≥5 min at 70°C (or 10 min at room temperature);

3. Add 300 μL of 96% ethanol for DNA precipitation, mix by brief vortexing;

4. Collect DNA by centrifugation (15,000× g) for 3 min;

5. Remove supernatant and wash the pellet with 500 μL 70% ethanol ;

6. Suspend in 30 to 50 μL water or TE to dissolve DNA;

7. Remove the debris by centrifugation (15,000× g for 1 min), and 1 μL supernatant was used for PCR.

**4. Yeast Transformation**

Competent cell preparation:

1. Activate strains. Cultures stored at -80 °C are streaked on YPDA medium plates and incubated at 30 °C for 2-4 days.
2. Pick the yeast single colony and draw a short line of 3-5mm on the YPDA medium plate, and incubate at 30 °C for 2-4 days.
3. When the yeast single colony grows to 2mm in diameter, the yeast cells are seeded into 3mLYPDA liquid medium and cultured overnight at 30 °C.
4. Transfer to a triangular flask containing 30-50 mLYPDA liquid medium the next day for further culture, wait for OD600 to reach 0.4-0.5, centrifuge at 3000 rpm for 5min, and discard the supernatant.
5. The pellet is suspended with 30-50mL of sterile deionized water. Centrifuge at 3000 rpm for 5 min and discard the supernatant.
6. The pellet was resuspended with 1.5mL of 1×LiAc (150μL of 10×LiAc Solution plus 1350μL of sterile water) and transferred to a 1.5mL centrifuge tube, centrifuged at 3000 rpm for 5min, and discarded supernatant.

   Note: 10× LiAc Solution is pH buffered and does not require the addition of TE as a buffer.
7. Add 1mL of 1× LiAc to resuspend, and the small volume transformation is divided according to 100μL per tube, and the library transformation is not divided.
8. Centrifuge at 3000 rpm for 5min, discard the supernatant, and the competent cells are prepared.

   Note: The prepared competent state is best used immediately and should not be left at room temperature for more than 5 h before centrifugation in step 8.

Conversion master mix formulation:

| Content | Volume |
| --- | --- |
| PEG Solution | 240μL |
| 10× LiAc Solution | 36μL |
| Carrier DNA | 10μL |
| Plasmid | 5 (≈200ng/μL) |
| Total | 360 (ddH20 padded) |

1. Add 360μL of the master mix to 1 competent cell, and repeatedly pipette the pellet to completely suspend the yeast cells in the master mix.
2. Incubate in a water bath at 30°C for 30 min and mix every 10 min.
3. (Add 20μLDMSO, optional) heat hit in a water bath at 42°C for 30min and mix well every 10min.
4. Centrifuge at 12,000 rpm for 15s and discard the supernatant.
5. (Optional step) Resuspend with 1 mL of YPD Plus Liquid Medium and incubate at 30°C shaker for 30-60 min. Centrifuge at 12,000 rpm for 15 s and discard the supernatant.
6. Add 0.1-1mL of sterile deionized water or 0.9% sodium chloride solution to resuspend the precipitate, coat screening medium plates, and incubate at 30 °C for 2-4 days.

**5. Delft Media Preparation**

This recipe is based on minimal media from Delft University (Verduyn 1992).

Further details can be found here:

Verduyn, C., Postma, E., Scheffers, W.A., Van Dijken, J.P., 1992. Effect of benzoic acid on metabolic fluxes in yeasts: A continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8, 501–517. doi:10.1002/yea.320080703

Delft minimal media for shake flask cultures without pH control

* 7.5 g/L (NH4)2SO4(ammonium sulfate)
* 14.4 g/L KH2PO4(monopotassium phosphate)
* 0.5 g/L MgSO4·7H2O(magnesium sulfate)
* pH adjusted to 6.5 or the pH needed

After autoclavation add:

* 2 mL/L trace metals solution
* 1 mL/L vitamins
* a certain amount of Glucose aqueous solution（20g/L）

**Trace metal solution：**

Total volume 1 L

1. Calibrate a pH electrode.
2. Add 800 mL MQ-H2O to a 1 L glass beaker. Add a magnetic stirrer.
3. Place the beaker on a stirrer with heating function and place the pH electrode in the water. Start stirring. The electrode should be kept in the solution during the whole preparation for a good result.
4. Add the chemicals listed below to the beaker, one by one. Set the pH to 6 after each addition. After the addition of EDTA the pH should be set to 4, the color will then turn to bright green.
   1. The chemicals should be added as carefully (slowly) as possible.
   2. pH is set with 2 M NaOH which is added dropwise. If you need to use HCl, use 0.1 M, 1 M or 2 M.
   3. Note: The first pH adjustment - after addition of FeSO4 - will take some time, be patient. However, you don’t have to wait until pH it is absolutely stable as long as the pH is around 6 when you add the next chemical.
   4. Before addition of EDTA heat the solution to hand warm (30-40˚C).

   | Name | Mass |
   | --- | --- |
   | FeSO4·7H2O | 3.0 g |
   | ZnSO4·7H2O | 4.5 g |
   | CaCl2·2H2O | 4.5 g |
   | MnCl2·4H2O | 1 g |
   | CoCl2·6H2O | 300 mg |
   | CuSO4·5H2O | 300 mg |
   | Na2MoO4·2H2O | 400 mg |
   | H3BO3 | 1 g |
   | KI | 100 mg |
   | Na2EDTA·2H2O | 19 g |
5. Remove the pH electrode. Cover the beaker with parafilm and then aluminum foil (light sensitive chemicals) and
   leave on a stirrer overnight. It will take time for the chemicals to dissolve completely.
6. In the morning adjust pH to 4 again. Adjust the volume to 1 L with MQ-H2O in a volumetric flask.
7. Autoclave the solution in one or several glass bottles. The solution will turn purple over time. Store at 4˚C.

**Vitamin solution:**

Vitamin solution 1000x (1 L)

Biotin is dissolved in 20 mL 0.1 M NaOH + approx. 700 mL MilliQ.

| Name | Mass |
| --- | --- |
| d-Biotin | 50 mg |

Adjust the pH to 6.5 with 1 M HCl, and add the following vitamins one by one:

| Name | Mass |
| --- | --- |
| D-Pantothenic acid hemicalcium salt | 1.0 g |
| Thiamin-HCl | 1.0 g |
| Pyridoxin-HCl | 1.0 g |
| Nicotinic acid | 1.0 g |
| 4-aminobenzoic acid | 0.2 g |

Adjust the pH to 6.5 (0.1 – 1 M NaOH), then add:

| Name | Mass |
| --- | --- |
| m-Inositol | 25 g |

Adjust the volume to 1.0 L and set the pH to 6.5. Sterilize by filtration and store in the cold. Fresh solution should be done every third month.

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}"In the realm of science, our experiment results are the brushstrokes on the canvas of discovery."{% endblock %}
{% block page\_content %}

#### Results

* [1. Exogenous addition](#part1)
* [2. Endogenous spermidine overexpression by CRISPR/Cas9](#part2)
* [3. Exogenous spermidine and thermospermine overexpression by plasmid](#part3)
* [4. Fine-tune exogenous thermospermine overexpression by plasmid from a comprehensive view](#part4)
* [5. Future Plan](#part5)

**Here, we present our most significant experimental findings, and the entire experimental procedure can be reviewed on the [Experiments](https://2023.igem.wiki/scu-bse-china/experiments). We commence by displaying a task table outlining our objectives and progress towards completion. Subsequently, we enumerate our pivotal results and future plan.**

| Number | Name | Description | Status |
| --- | --- | --- | --- |
| 1 | Exogenous addition | We cultured wild-type strain in Delft(with uracil) media containing varying concentrations of spermidine gradients to assess their thermo-tolerance capabilities. | √ |
| 2 | Endogenous spermidine overexpression by CRISPR/Cas9 | We utilized CRISPR/Cas9 technology to construct yeast strains with spermidine or thermospermine overexpression and assessed their thermo-tolerance capabilities. | √ |
| 3 | Exogenous spermidine and thermospermine overexpression by plasmid | We utilized plasmid-based exogenous overexpression to introduce spermidine (SPE1) and thermospermine (AtACL5) in the OAZ1 knockout strain and subsequently assessed their thermo-tolerance capabilities. | √ |
| 4 | Fine-tune exogenous thermospermine overexpression by plasmid from a comprehensive view | We fine-tuned the expression levels of SPE2, SPE3, and AtACL5 by plasmid in the OAZ1Δ::SPE1 strain and subsequently assessed their thermo-tolerance capabilities. | ... |

**1. Exogenous addition**

The hypothesis of our study is that polyamines can enhance the thermo-tolerance of *S. cerevisiae*. Therefore, we initially conducted exogenous spermidine addition experiments to preliminarily investigate the feasibility of our hypothesis.

We established different experimental groups with final spermidine concentrations ranging from 1nM to 1mM. After a 48-hour culturing at 38°C, we measured the growth of the wild-type strain (Figure 1).

![figure1.png](https://static.igem.wiki/teams/4937/wiki/results/p1.png)

Figure 1

The results indicated that spermidine did not significantly enhance the thermo-tolerance of the strain. This could be attributed to several potential factors. We have summarized the two primary possibilities: Firstly, if our hypothesis is correct, it is plausible that spermidine may not be able to enter the cell and thus fails to exert its effects. Moreover, extracellular spermidine may not impact cell growth. Alternatively, our hypothesis may not hold true, implying that spermidine cannot enhance the cell's thermo-tolerance capabilities.

**2. Endogenous spermidine overexpression by CRISPR/Cas9**

According to literature reports, the presence of the endogenous OAZ1 gene in *S. cerevisiae* restricts the synthesis of polyamines, thereby limiting the direct precursor spermidine's production. To enhance the metabolic flux of spermidine synthesis in our strain, we initiated the knockout experiment of the endogenous OAZ1 gene. Our experiment consisted of three main phases: fragment construction, yeast transformation, and validation.

We obtained the plasmid backbone through PCR amplification, as shown in Figure 2A, and its size was as expected. Subsequently, we annealed to obtain the gRNA sequence. Following the extraction of yeast genomic DNA, we amplified the upstream and downstream sequences of the repair template by PCR, as depicted in Figure 2B, and their sizes were as expected. Finally, we generated the complete repair template through fusion-PCR, as illustrated in Figure 2C, and its size was as expected.

![figure1.png](https://static.igem.wiki/teams/4937/wiki/results/p2.png)

Figure 2

Following the acquisition of the aforementioned fragments, we proceeded with yeast competent cell preparation and transformation onto Delft agar plates. Subsequently, we randomly selected 10 individual clones from the plates for further expansion culture and genomic DNA extraction for preliminary validation. The length of PCR at the original locus was 1949bp, as shown in the Positive Control (PC) group in Figure 3A. The theoretically expected length after successful knockout should be 1011bp. Gel electrophoresis results indicated that colony 1-5 might be yeast strains with the OAZ1 gene knockout, as shown in Figure 3A. Therefore, we sequenced the PCR products from these colonies, and the results aligned with our expectations (Figure 3B and 3C).

![figure1.png](https://static.igem.wiki/teams/4937/wiki/results/p3.png)

Figure 3

SPE1 encodes an ornithine decarboxylase, catalyzing the decarboxylation of ornithine to form putrescine, and its activity is inhibited by the antizyme enzyme OAZ1. To enhance putrescine expression and consequently increase the supply of spermidine precursors, we constructed yeast strains with OAZ1 knockout and SPE1 knock-in for putrescine (and subsequently spermidine) overexpression. The basic experimental procedure is similar to the one described above.

After extracting yeast genomic DNA, we amplified the upstream and downstream sequences of the repair template by PCR, as well as the TEF1p, PRM9t (Figure 4A) and SPE1 (Figure 4B) fragments, and their sizes were as expected. Subsequently, we successfully prepared the repair template using a two-round fusion-PCR strategy. In the first round, we fused the first two fragments (upstream-2 and TEF1p) and the last two fragments (PRM9t and downstream-2), as shown in Figure 4C. In the second round, we fused three fragments (upstream-2-TEF1p, SPE1, and PRM9t-downstream-2), as shown in Figure 4D.

![figure1.png](https://static.igem.wiki/teams/4937/wiki/results/p4.png)

Figure 4

Then we tried to transform the fragments and validate the colonies. The length at the original locus was 1949bp, as shown in the Positive Control (PC) group in Figure 5A. The theoretically expected length after successful editing should be 3080bp. Gel electrophoresis results indicated that Colony2-1, Colony2-3, Colony2-4, and Colony2-7 might be successfully edited strains (Figure 5A). Therefore, we selected the PCR product from Colony2-1 for sequencing, and the results matched our expectations (Figure 5B).

![figure1.png](https://static.igem.wiki/teams/4937/wiki/results/p5.png)

Figure 5

Then we employed 5-FOA for counter-selection to obtain strains that had lost the gRNA editing plasmid.

And we performed HPLC test to ensure that our engineered strains have improved production of putrescine and/or spermidine. As shown in Figure 6, the OAZ1Δ strain produces 5.70 mg/L putrescine and 7.15 mg/L spermidine, and the OAZ1::SPE1 strain produces 9.5 mg/L putrescine and 15.2 mg/L spermidine. Compared to wild type strain produces 1.3 mg/L putrescine and 2.9 mg/L spermidine, the both engineered strain has a higher production of polyamines, which means that we successfully increased the flux of polyamine synthesis.

![figure1.png](https://static.igem.wiki/teams/4937/wiki/result-new/p6.png)

Figure 6

Subsequently, we conducted a thermo-tolerance growth test at 35°C for the aforementioned strains, as shown in Figure 7.

![figure1.png](https://static.igem.wiki/teams/4937/wiki/result-new/p7.png)

Figure 7

The experimental results indicated that the knockout of the OAZ1 gene and the overexpression of SPE1 did not have a significant impact on the thermo-tolerance of the strains.

After reaching this conclusion, our subsequent experiments will focus more on putrescine rather than its precursors (i.e., ornithine and spermidine). The *At*ACL5 gene, originating from Arabidopsis thaliana, encodes a thermospermine synthase capable of catalyzing the aminopropylation of dSAM to form thermospermine. We plan to construct a repair template with the sequence OAZ1\_upstream-TEF1p-SPE1-PRM9t-TDH3p-*At*ACL5-DIT1t-OAZ1\_downstream to simultaneously knockout OAZ1 and knock-in SPE1 and *At*ACL5 to achieve thermospermine overexpression.

We initially obtained the short fragment (Figure 8A and 8B) through PCR. Subsequently, we amplified the SPE1 fragment (Figure 8C) from the previously used repair template. Following that, we obtained the AtACL5 fragment (Figure 8D) through fusion PCR. Finally, we acquired the complete repair template (Figure 7E) through fusion PCR.

![figure1.png](https://static.igem.wiki/teams/4937/wiki/result-new/p8.png)

Figure 8

However, numerous small colonies grew on the Delft plates after yeast transformation, as shown in Figure 9. These colonies could not be expanded in Delft media, and as a result, we never obtained positive clones. We suspect that this may be due to low transformation efficiency leading to a low positivity rate. Additionally, the use of uracil-containing medium during the preparation of competent cells and post-transformation recovery may have caused some cells that were not successfully transformed to maintain uracil for a period, allowing them to grow and divide, thereby leading to false positives.

![figure1.png](https://static.igem.wiki/teams/4937/wiki/result-new/p9.png)

Figure 9

Subsequently, we improved the transformation process to enhance its efficiency, as detailed in [Engineering](https://2023.igem.wiki/scu-bse-china/engineering). Additionally, when fusing multiple short fragments into longer ones, we refined and optimized the details of fusion PCR, significantly increasing its efficiency, as elaborated in [Engineering](https://2023.igem.wiki/scu-bse-china/engineering).

**3. Exogenous spermidine and thermospermine overexpression by plasmid**

Using the OAZ1 knockout strain we previously constructed, we generated yeast expression plasmids to overexpress SPE1, *At*ACL5, as well as both SPE1 and *At*ACL5, to achieve overexpression of putrescine (and subsequently spermidine) and thermospermine. We initially obtained the plasmid backbone through PCR (Figure 10: 7-1 to 7-4) and the insert fragments (Figure 10: 7-5 to 7-7; 7-5 is TEF1p-SPE1-PRM9t, 7-6 is TDH3p-*At*ACL5-DIT1t, 7-7 is TEF1p-SPE1-PRM9t-TDH3p-*At*ACL5-DIT1t). Subsequently, we used homologous recombination to construct the complete plasmids and validated their successful construction through sequencing (Figure 12).

![figure1.png](https://static.igem.wiki/teams/4937/wiki/results/p9.png)

Figure 10

![figure1.png](https://static.igem.wiki/teams/4937/wiki/results/p10.png)

Figure 11

And we performed HPLC test to ensure that our engineered strains have improved production of putrescine, spermidine and thermospermine. As shown in figure 12, the strain contains plasmid that overexpress SPE1 has a higher production of putrescine and spermidine, namely 6.95 mg/L and 8.1 mg/L, respectively, compared to 5.00 mg/L and 6.7 mg/L in strain contains plasmid vector. And the strain contains plasmid that overexpress SPE1 and AtACL5 produces 5.2 mg/L spermidine, less than negative control group. As we didn’t get the standard of thermospermine, we didn’t get a directly evidence of thermospermine prodution. However, according to the decreased spermidine, we think it may indicated the improvement production of thermospermine.

![figure1.png](https://static.igem.wiki/teams/4937/wiki/result-new/p12.png)

Figure 12

Then we tested the growth of these strains under 35°C conditions (Figure 13). The results indicate that the expression of the *At*ACL5 gene did not have a significant impact on the yeast's thermo-tolerance capabilities.

![figure1.png](https://static.igem.wiki/teams/4937/wiki/results/p11.png)

Figure 13

**4. Fine-tune exogenous thermospermine overexpression by plasmid from a comprehensive view**

Furthermore, after conducting additional literature research, we discovered that the expression of thermospermine not only depends on the level of putrescine but also on the level of dSAM (decarboxylated S-adenosylmethionine). And then we drew the detailed picture of polyamine synthesis in yeast(Figure 14). We hypothesized that even though we overexpressed the genes for putrescine and thermospermine, the insufficient intracellular dSAM level might have resulted in very low thermospermine expression. Additionally, the strong competitive nature of spermidine and thermospermine synthesis for the same precursors further reduced thermospermine levels.

![figure1.png](https://static.igem.wiki/teams/4937/wiki/result-new/p14.png)

Figure 14. Polyamine synthesis in yeast.

(The red arrow means the increased flux, and the blue color means the decreased flux, and the green arrow means a consumption process of our core substrate. The solid arrow means a direct reaction and the dotted arrow means a multi-steps reaction.)

Therefore, we plan to construct strains with simultaneous overexpression of dSAM to increase the levels of thermospermine. We designed a plasmid library to stably express SPE2 and tune the expression level of SPE3 and *At*ACL5 to fully use the spermidine substrate and examine the numerical relationship between spermidine and thermospermine.(Figure 15)

![figure1.png](https://static.igem.wiki/teams/4937/wiki/result-new/p15.png)

Figure 15

We firstly achieved all short single fragment by PCR from the genome of *S. cerevisiae* and plasmid(Figure 16). Then we connected all single expression device by fusion PCR(Figure 17).

![figure1.png](https://static.igem.wiki/teams/4937/wiki/result-new/p16.png)

Figure 16

![figure1.png](https://static.igem.wiki/teams/4937/wiki/result-new/p17.png)

Figure 17

However, the Gibson coloning results were not as expected. We performed the double enzyme digestion experiment and PCR to check whether the sequence of whole plasmids are right. And only one colony shows expected results(Figure 18), we guessed it may come from the unexpected similarity of homologous fragment. Due to time limited, we didn’t complete this part of work. However, we will keep on trying this design.

![figure1.png](https://static.igem.wiki/teams/4937/wiki/result-new/p18.png)

Figure 18

**5. Future Plan**

Though this season is end, our project keeps going on. We have many ideas about this project.

Firstly, the endogenous metabolism is important for any organism, which means any alteration should consider the negative effect on strain itself. In our project, we overlooked the flux from ornithine to arginine, and this may cause a decreased arginine production, which is harmful to our engineered strain. So, we plan to increase the production of ornithine to provided enough substrate for polyamine synthesis.

Secondly, not only thermospermine, as the information we collected from different articles, there are many kinds of long-chain polyamines and their derivatives may have an important effect on the thermo-tolerance of yeast strain. These unusual polyamines are our main targets for future.

Thirdly, polyamines play an important role in different kinds of stress-tolerance. We will use our strain to test whether it has other stress-tolerance, such as the tolerance to extreme pH, toxic product and so on.

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