Engineering Success

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:

Process:

②PCR with primers:

Process:

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.

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.



• 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.



• 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 for reference and dissemination.

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).



• 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).



• 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.

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).



• 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 reported¹.
• Test: After constructing the strain, we performed HPLC test to examine the production of putrescine and spermidine(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.