We designed several parts for testing localization of different sequences in S. cerevisiae:

• MTS1 (mitochondrial targeting sequence 1). The sequence was adapted from [1]. We registered this sequence in the Parts Registry under BBa_K5054002
• uTP1 and uTP2 (UCYN-A transit peptide). The sequences for these were designed by us through bioinformatics analysis of the native UCYN-A imported proteins (see Materials and Methods and Results for more details). These two sequences were submitted to the Parts Registry under BBa_K5054000 and BBa_K5054001 respectively.
  uTP1 Amino Acid Sequence

  WLEEWRERLECWWGPVGTQTQLGACMGALGLHLGSRLDNEQETQTISAIVAEPGCEWVEEAAPGLPDFPEPFSLPPIPRL

  uTP2 Amino Acid Sequence

  WLEEWRERLECWWLDPKTQTQLGACMGALGLHLGSRLDIAPYFAWRAALLGRAPPPSARAEPGCEWVEEALDDLPDFPEPFSLPPIPRL

These parts were inserted into the plasmid pUDE1311, designed for yeast expression, descibed in detail in [2]. This plasmid consists of a yeast-optimized NeonGreen fluorescent protein gene under control of the S. cerevisiae HHF2 promoter, with a URA3 selectable marker for yeast and ampicillin resistance for bacterial selection, carried on a high-copy 2-micron vector.

The original sequence of the pUDE1311 plasmid is available for download here.

Primers were designed to insert MTS1 at the N-terminal end of mNeonGreen and uTP1/2 at the C-terminal. A 6X His tag was added to the insert, in between mNeonGreen and uTP to aid in purification later on. For more details see Materials and Methods and Results.

The sequences for the parts can be found in the Parts Registry. Sequences for the parts inserted into the plasmid can be downloaded here: MTS1, uTP1, uTP2.

Similar to yeast, several parts were designed to test uTP and other targeting sequences in C. reinhardtii. In this case, we also wanted to compare uTP's behavior to chloroplast localizing proteins, since UCYN-A shares its native host with a chloroplast, so it is possible that they interact.

The following inserts were created:

• mtTP70C mitochondrial transit peptide adapted from the Parts Registr BBa_K4806011 by RPTU-Kaiserslautern 2023.
• CTP_PsaD chloroplast transit peptide adapted from the Parts Registry BBa_K4806014 by RPTU-Kaiserslautern 2023.
• uTP1 and uTP2 with sequences identical to the yeast parts.

These parts were inserted into the plasmid pOpt2, optimized for C. reinhardtii expression. The plasmid is described in detail [3]. This plasmid consists of an mVenus fluorescent protein gene containing the RBCS2 intron 2, flanked by HSP70A promoter and RBCS2 promoter sequences on both sides, with a selectable marker (ble) containing an intron, and XbaI and KpnI restriction sites at the ends for cloning purposes.

The original sequence of the pOpt2 plasmid is available for download here.

Primers were designed to insert mtTP70C and CTP_PsaD at the N-terminal end of mVenus and uTP1/2 at the C-terminal. For more details see Materials and Methods and Results.

The sequences for the parts can be found in the Parts Registry. Sequences for the parts inserted into the plasmid can be downloaded here: mtTP70C, CTP_PsaD, uTP1, uTP2.

1. Dong, C., Shi, Z., Huang, L., Zhao, H., Xu, Z., & Lian, J. (2021). Cloning and characterization of a panel of mitochondrial targeting sequences for compartmentalization engineering in Saccharomyces cerevisiae. Biotechnology and Bioengineering, 118(11), 4269–4277. https://doi.org/10.1002/bit.27896
2. Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology, 4(9), 975–986. https://doi.org/10.1021/sb500366v
3. Lauersen, K. J., Kruse, O., & Mussgnug, J. H. (2015). Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit. 99(8), 3491–3503. https://doi.org/10.1007/s00253-014-6354-7