Plasmids used in this study are listed in Table 1. Restriction enzymes, like BamHI and BbsI, 10X T4 ligation buffer, T4 polynucleotide kinase (PNK), T4 ligase and HiFi DNA Assembly Master Mix for Gibson assembly were all bought from New England Biolabs and were used according to the manufacturer’s protocol. The constructed plasmid products were transformed into homemade TOP10 or Stable3 competent cells according to the protocol written below.
Plasmid was extracted from subcultured E. coli through ZR Plasmid Miniprep Classic (Zymo Research) according to the manufacturer’s protocol. The extracted plasmid underwent Sanger sequencing conducted by Academia Sinica Core Facility.
The plasmid pSL122 was constructed through Gibson assembly of two PCR amplicons. One was amplified from pET28a with primers SL410 and SL412. The other amplicon was amplified from plasmid mito-EGFP with primers SL411 and SL413. The Gibson assembly was conducted by mixing 1 µL HiFi DNA Assembly Master Mix and 0.5 µL of each amplicon and heated at 50°C for 1 hour. The product was transformed into homemade Stable3 competent cell.
The plasmid pSL127 was constructed through Gibson assembly of three PCR amplicons. All
SL435 and SL436, or SL437 and SL438. The Gibson assembly was conducted by mixing 1 µL HiFi DNA Assembly Master Mix and 0.33 µL of each amplicon and heated at 50°C for 1 hour.
The product was transformed into homemade Stable3 competent cell. The ligation was conducted by
The plasmids pSL127B to pSL127G were all constructed through Gibson assembly of two PCR amplicons. One of the two amplicons is the same for all the six plasmids and was amplified from pSL127 with primers SL448 and SL449. The other amplicons are different but were all amplified from a commercially synthesized gBlock gene fragment SL533 from IDT with various primer pairs. For pSL127B, primers SL456 and SL457 were used to amplify DNA segment of double repeats of COX8A MTS. For pSL127C, primers SL460 and SL461 were used to amplify DNA segment of S. cerevisiae COX4P MTS. For pSL127D, primers SL450 and SL451 were used to amplify DNA segment of ACACB optimized MTS. For pSL127E, primers SL454 and SL455 were used to amplify DNA segment of GLDC optimized MTS. For pSL127F, primers SL452 and SL453 were used to amplify DNA segment of POLG optimized MTS. For pSL127g, primers SL458 and SL459 were used to amplify DNA segment of MTHFD1L optimized MTS.
The Gibson assembly was conducted by mixing 1 µL HiFi DNA Assembly Master Mix and 0.5 µL of each amplicon and heated at 50°C for 1 hour. The product was transformed into homemade Stable3 competent cell.
The plasmids pSL127Ga, pSL127Gb and pSL127Ge were generated through “round-the-horn” technique. Firstly, pSL127G was amplified with primer pairs SL510 and SL509, SL512 and SL511, or SL601 and SL600, respectively. Next, the PCR amplicons were ligated to form the final product. The ligation mixture included 2.5 µL linear PCR product, 0.5 µL molecular ddH2O, 0.5 µL 1 mM ATP, 10x T4 ligation buffer, T4 PNK and T4 ligase. The mixture was
incubated at 37°C for 1 hour. The product was transformed into homemade Stable3 competent cell.
The plasmids pSL127Gc, pSL127Gd and pSL127Gf were generated through Gibson assembly from pSL127Ga, pSL127Gb and pSL127Ge, respectively. In general, pSL127Ga, pSL127Gb and pSL127Ge were all amplified with primer pairs SL563 and SL562 as backbones.
On the other hand, the insert sequence for the three constructs was the same one which was amplified from SL533 gBlock (IDT) with primer pairs SL551 and SL552. Finally, the Gibson assembly was conducted by mixing 1 µL HiFi DNA Assembly Master Mix, 0.5 µL backbone PCR product and 0.5 µL insert PCR product and heated at 50°C for 1 hour. The product was transformed into homemade Stable3 competent cell.
The plasmids pSL146, pSL147, pSL148, pSL149, pSL187 and pSL188 were generated through ligation of BbsI-digesed pSL127Ga, pSL127Gb, pSL127Gc, pSL127Gd, pSL127Ge and pSL127Gf, respectively, with annealed oligos. For pSL146 and pSL148, the annealed oligos was SL584 and SL585. For pSL147 and pSL149, the annealed oligos was SL586 and SL587. For pSL187 and pSL188, the annealed oligos was MF3 and MF4.
The plasmids pSL245 was generated through “round-the-horn” technique by amplifying pSL146 with primers MF44 and MF45. After mixing 2.5 µL linear PCR product, 0.5 µL molecular ddH2O, 0.5 µL 1 mM ATP, 0.5 µL 10x T4 ligation buffer, 0.5 µL T4 PNK and 0.5 µL T4 ligase, the mixture was incubated at 37°C for 1 hour. The product was transformed into homemade TOP10 competent cell. Similarly, the plasmid pSL297 was also generated through
“round-the-horn” technique by amplifying pSL245 with primers MF94 and MF95. The ligated product was transformed into homemade TOP10 competent cell as well.
The plasmids pSL246 to pSL249 and pSL298 were generated through “round-the-horn”
technique by amplifying pSL179 with various primer pairs. For pSL246, primers MF47 and MF48 were used. For pSL247, primers MF49 and MF50 were used. For pSL248, primers MF51 and MF52 were used. For pSL249, primers MF53 and MF54 were used. For pSL298, primers MF96 and MF97 were used. After mixing 2.5 µL linear PCR product, 0.5 µL molecular ddH2O, 0.5 µL 1 mM ATP, 0.5 µL 10x T4 ligation buffer, 0.5 µL T4 PNK and 0.5 µL T4 ligase, the mixture was incubated at 37°C for 1 hour. The product was transformed into homemade TOP10 competent cell.