Contribution

In yeast engineering, the construction of simple gene expression circuits typically involves the arrangement of a promoter-CDS-terminator module. Certain frequently employed high-expression parts, such as GDH3p and TEF1p, experience repetitive utilization during yeast engineering endeavors. To enable the seamless integration of distinct promoters, CDSs, and terminators into cohesive expression modules, a fusion PCR approach is often employed. A conventional fusion PCR strategy needs the incorporation of approximately 20bp of homologous sequences upstream and downstream of the three constituent parts during PCR amplification (see Figure 1). Nonetheless, this practice mandates the use of CDS-specific primers whenever the same promoter or terminator is employed, thus proving both inefficient and unsuitable for high-throughput experiments.

In this context, we have devised an economical and useful PCR strategy (see Figure 2). Specifically, we refrain from appending homologous sequences to reusable parts such as promoters and terminators. Instead, we introduce slightly longer homologous sequences (approximately 40bp) exclusively to the ends of parts with comparatively lower utilization frequencies, such as CDSs. Through testing, we have proved the comparable efficacy of this methodology.

Fusion PCR, a pivotal technique in strain engineering, facilitates the assembly of multiple DNA fragments into a unified construct. In our investigations, we evaluated the performance of two frequently employed enzymes, namely KOD OneTM PCR Master Mix and PrimeSTAR Max Premix(2×), for fusion PCR. Remarkably, we observed PrimeSTAR Max Premix(2×) to exhibit exceptional efficiency within the context of fusion PCR. Consequently, we have compiled a comprehensive methodology for fusion PCR utilizing PrimeSTAR Max Premix(2×), tailored to accommodate various fragment numbers. We intend for this protocol to serve as a valuable resource for forthcoming iGEM teams seeking guidance in their endeavors.

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 ºC for 3 min, 15 cycles of 98 ºC for 10 s, 58 ºC 15 s, 72 ºC for 1 min/kb, at last 72 ºC 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 ºC for 3 min, 30 cycles of 98 ºC for 10 s, 56 ºC 15 s, 72 ºC for 1 min/kb, at last 72 ºC 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 ºC 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.

For the fusion of up to five DNA fragments (as illustrated in Figure 3), a direct five-fragment fusion PCR strategy was implemented. Employing this approach, we achieved successful assembly of the extended fragment upstream-TEF1p-SPE1-PRM9t-downstream during the construction of the ΔOaz1::SPE1 strain, as depicted in Figure 6. The acquisition of individual fragments is outlined in Figures 4 and 5.

For fusion PCR involving more than five fragments, we recommend employing a multi-step approach for optimal efficiency. As illustrated in Figure 7, here is an example of eight fragments. The process begins with the fusion PCR of the first four fragments and the last four fragments separately. Subsequently, two four-fragment fusion PCRs were performed. This step-wise fusion PCR strategy ensures maximal efficiency.

We successfully applied this methodology during the construction of the ΔOaz1::SPE1-AtACL5 strain, resulting in the assembly of the extended fragment upstream-TEF1p-SPE1-PRM9t-TDH3p-AtACL5-DIT1t-downstream, as demonstrated in Figure 8. The acquisition of individual fragments is detailed in Figures 4, 5, 9, and 10.

We contributed the standard experiment protocol for polyamine detection for future iGEM team who want to check the content of polyamine. And this can be found in Experiment.

We contributed lots of parts related to yeast engineering and polyamine synthesis, which includes 15 basic parts and 32 composite parts. And this can be found in Parts.