UCYN-A enriched proteins were selected from a protein quantification dataset [26]. Multiple sequence alignment was performed on these proteins using COBALT [15] with default parameters, followed by alignment cleanup using CIAlign [16] with the remove-insertions option. Alignments exhibiting at least 60% coverage in the 880-1010 region were selected for further analysis.

The Gblocks [castresana] software was employed to crop the selected alignments, with a minimum of 104 sequences for both conserved and flank positions, a maximum of 15 contiguous nonconserved positions, a minimum block length of 10, and allowing all gap positions. MEME [18] was subsequently run on the cropped alignments using protein mode, with 10 motifs requested and a negative dataset, employing the differential enrichment objective function.

Motif analysis involved computing the positions of each motif's occurrences relative to motif #1, which was present in 184 out of 204 sequences. The distribution of these relative positions was visualized, along with the different combinations of motifs and their relative positions within these combinations.

The relationship between the mature domain and the motifs in the corresponding transit peptide was analyzed using embeddings computed with the prot_t5_xl_uniref50 [19] model, obtained from HuggingFace Model Hub. Logistic regression, decision tree, random forest, and support vector machine classifiers, as implemented by scikit-learn [20] using default parameters, were trained to predict motif combinations based on protein embeddings and amino acid contents of the mature domain. The performance of classifiers was tested using 5-fold cross-validation. The statistical significance of classifier accuracy was evaluated using permutation tests [21], as implemented by scikit-learn’s permutation_test_score, with a significance threshold of p < 0.05.

The design of fluorescent uTP constructs was guided by a previously trained logistic regression classifier, which predicted the most probable motif combinations for each sequence. To more accurately mimic the native protein structure, the spatial arrangement of motifs was carefully considered. In native proteins, motifs are typically separated by short intervening sequences rather than occurring in direct succession. To replicate this natural spacing in the constructs, gaps between motifs were incorporated. These gaps were populated with amino acids randomly selected based on the frequency distribution observed in the corresponding native protein sequences.

For structural analysis, AlphaFold3 [22] was used to predict structures for each UCYN-A imported protein with default parameters. The resulting structures were analyzed using Biopython's PDB module [23]. A subset of each structure with strong C-terminal sequence similarity was selected for further analysis. These structures were aligned using PyMOL's [schroedinger] cealign command, and the alignments were used to create a consensus structure by averaging the position of the aligned residues from each structure.

To evaluate the predicted fluorescent uTP constructs, their structure was predicted using AlphaFold3 and aligned onto the consensus structure found earlier using PyMOL’s cealign command.

Protein localization was predicted for each UCYN-A enriched protein using MuLocDeep [25]. The attention weights as a function of sequence position were visualized, and the total C-terminal and N-terminal attention weights were compared across different localizations.

Using raw RNA-seq data from [27], a transcriptome assembly was constructed using DRAP [35] with the oases assembler and default parameters. The completeness of the assembly was tested with BUSCO [33]. Proteins were predicted from the transcriptome assembly with TransDecoder [34]. Ortholog analysis was performed using OrthoFinder [36]. See Data and Code for the predicted proteome.

Plasmids were designed to express fluorescent proteins fused to various targeting sequences in Saccharomyces cerevisiae and Chlamydomonas reinhardtii. These sequences included uTP1/2 (UCYN-A transit peptide, identified through bioinformatics analysis), MTS1 (mitochondrial targeting sequence 1) [13], mTP (mitochondrial transit peptide)[38] and cTP (chloroplast transit peptide) [14]. The uTP1/2 sequence was inserted at the C-terminal end of the fluorescent proteins, while MTS1, mTP and cTP were incorporated at the N-terminal end. For uTP1/2 constructs, a His6-tag was incorporated between the transit peptide and the fluorescent protein to facilitate future purification experiments.

Two existing vectors with fluorescent proteins were used as backbones for this study. For S. cerevisiae, we utilized pUDE1311 [11], which contains mNeonGreen and a URA3 marker for selection (full genotype: ConLS-ScHHF2p-ymNeonGreen-ScENO1t-ConR1-URA3-2µ-AmpR). This vector was kindly provided by Marcel Vieira Lara of TU Delft. For C. reinhardtii, we used pOpt2 [9] [10], containing mVenus and a ble marker for selection. This plasmid, kindly provided by Friedrich Kleiner from the MEA lab at TU Delft, also features specific introns in the mVenus coding sequence to prevent gene silencing. Both plasmids contain an ampicillin resistance gene for selection in E. coli. The complete sequences of both unmodified vectors and plasmids with all inserts have been made publicly available [link].

All plasmids, inserts, and primers were designed in SnapGene (www.snapgene.com). The inserts containing the transit peptides were synthesized by GenScript (Piscataway, NJ, USA). Gibson Assembly was employed for plasmid construction. Primers with specific overhangs were designed for PCR amplification of inserts and linearization of plasmid backbones at both the C-terminal and N-terminal regions, for inserting uTP1/2 and MTS1/mTP/cTP respectively.

PCR amplification was performed using KOD polymerase. To eliminate leftover circular plasmid from the backbone amplification process, DpnI digestion was utilized. Gibson Assembly was then carried out to combine the linearized plasmid backbones with the uTP, MTS1, mTP and cTP inserts using the Gibson Assembly® Cloning Kit by New England Biolabs [1], following the manufacturer’s protocol [2]. pUC19 and a DNA fragment provided by the kit were used as positive controls.

See Parts and Sequences for more details on the design and sequence of our parts.

5-alpha competent E. coli cells from New England Biolabs Gibson Assembly® Cloning Kit were transformed with the assembled plasmids and the positive control using the manufacturer’s protocol for chemical transformation [3].

Transformed cells were plated on LB (lysogeny broth) agar containing ampicillin (50 µg/mL). Colony PCR targeting the inserts was conducted on the resulting E. coli colonies to verify correct insert incorporation.

Plasmids were isolated from selected and verified E. coli colonies using the PureYield™ Plasmid Miniprep System (Promega, Madison, WI, USA), following the manufacturer's protocol [miniprep_protocol] with slight modifications. Specifically, the elution step was performed using pre-warmed nuclease-free water (37°C) and the incubation time was extended to 10 minutes to increase yield. Plasmid purity and concentration were subsequently assessed using a NanoDrop™ Microvolume Spectrophotometer.

S. cerevisiae CEN.PK113-5D (full genome available at [12]), kindly provided by Marcel Vieira Lara of TU Delft, was cultured on YPD (yeast extract peptone dextrose) media. This particular strain carries a mutation in the URA3 gene, rendering it auxotrophic for uracil. This auxotrophy was used as a selective marker in subsequent transformation experiments.

S. cerevisiae was chemically transformed with plasmids encoding the fluorescent protein mNeonGreen tagged with either the mitochondrial transit peptide (MTS1) or UCYN-A transit peptide (uTP2), alongside a control without transit peptide (mNeonGreen only) and negative control (no plasmid) (see Protocols). Transformed cells were plated on uracil-deficient media to select successful transformants.

Colony PCR (see Protocols) was performed on the formed E. coli colonies, targeting the inserts to confirm the presence of the transit peptide sequences. Positive colonies were inoculated in liquid selective media for plasmid amplification and isolation.

Verification of isolated plasmids was performed via whole-plasmid sequencing done by Plasmidsaurus Inc. (Pasadena, CA, USA) using Oxford Nanopore Technologies long-read sequencing. The results were interpreted in SnapGene software (www.snapgene.com) by aligning the consensus sequence generated from sequencing with the expected plasmid.

S. cerevisiae colonies that passed the screening were inoculated in liquid selective medium and examined under a fluorescent spinning disk confocal microscope (Nikon Eclipse Ti-2, Crest X-light V3, 488-nm laser, FI emission filter) using a 100x oil-immersion objective to investigate sub-cellular localization of expressed fluorescent proteins.

The UVM4 strain of C. reinhardtii [31] was selected for transformation due to its higher transformation efficiency compared to more common strains. The strain was kindly provided by Dr. Ralph Bock from the Max Planck Institute of Molecular Plant Physiology. Zeocin was used for selection as this strain carries no resistance to it and our plasmids contained the ble gene conferring resistance to successful transformants.

Transformation of C. reinhardtii is more efficient with small linear DNA than with larger and circular DNA [9]. Therefore, before transformation, plasmid constructs were digested with KpnI and XbaI restriction enzymes. Then, we attempted to excise the bands with the expected length (... bp) for the TP-mVenus-ble expression cassette from the gel, followed by column purification. However, the DNA yield after purification was very low. Therefore, we then used an ethanol precipitation method (see protocols) to purify the DNA. As a result, the purified DNA solution contained the TP-mVenus-ble expression cassette as well as the vector backbone.

For the transformation of C.reinhardtii cells, a protocol adopted from [30] and [37] was used (see Protocols). C. reinhardtii was transformed using electroporation with an exponential decay pulse. The digested plasmids were used containing fluorescent protein constructs with an antibiotic marker (mVenus-ble) tagged with either the mitochondrial transit peptide, chloroplast transit peptide, or UCYN-A transit peptide (uTP2), alongside a control without transit peptide (mVenus-ble only) and a negative control (no plasmid) (see Protocols). Transformed cells were plated on TAP agar media containing zeocin to select successful transformants.

A protocol for fusing E. coli into S. cerevisiae using polyethylene glycol (PEG) was adapted from [6]. E. coli NCM3722 expressing fluorescent reporter PlsB-msGFP2 (plasmid carrying GFP available here: [plsb-mgfp2]), kindly provided by Jaïrus Beije from TU Delft, was used to track fusion success under microscopy.

The E. coli cells were grown to OD600 ~0.8, harvested, and resuspended in bacterial resuspension buffer. Yeast cells were converted to spheroplasts using zymolyase treatment. The E. coli suspension was mixed with sorbitol and immediately added to the spheroplast suspension. After incubation, the mixture was combined with PEG buffer. Following centrifugation, the pellet was resuspended in YPDS (yeast extract peptone dextrose sorbitol) medium and incubated.

Control samples were also prepared: S. cerevisiae treated with PEG but without E. coli and S. cerevisiae with E. coli but without PEG treatment.

All samples were imaged using fluorescent scanning confocal microscopy (Nikon confocal A1R, excitation 488-nm@16mW, emission filter 525/50) to evaluate fusion events. Cells were segmented using the transmitted light channel and instances of bright fluorescent spots within cells were evaluated. Image analysis was performed in ImageJ.

We are making our code and data publicly available to help future iGEM teams who wish to build on our work.

The code used to characterize the uTP sequences is available on this repository.

The list of selected B. bigelowii protein sequences with C-terminal uTP can be downloaded here

The consensus structure obrained for uTP can be downloaded here

Our predicted B. bigelowii proteome is available upon request.

The sequences of all primers used in our experiments can be found here here

See Parts and Sequences for more details on the design and sequence of our parts.

The lab notebook can be viewed here.

Adapted from [7] following [8].

• YPD (yeast extract peptone dextrose) liquid media
• H₂O (sterile)
• Polyethylene glycol 3350 (PEG 3350; 50% w/v)
• TE buffer (pH 8.0)
• Single-stranded carrier DNA (2mg/ml)
• 1.0M Lithium acetate stock solution (LiAc)
• Plasmid DNA and yeast cells

• Spectrophotometer
• Water baths (30°C, 42°C, 96°C)
• Shaking incubator
• Centrifuge
• Vortex mixer
• Micropipettes and sterile tips
• Microfuge tubes and 50ml centrifuge tubes

1. Inoculate yeast cells in 100ml YPD and grow overnight at 30°C, 200rpm.
2. After 12-16h, measure culture density:
• Dilute 10μl cells in 1ml water
• Measure OD₆₆₀ (0.1 OD₆₆₀ = 1x10⁶ cells/ml)
3. Add 2.5x10⁸ cells to 50ml pre-warmed YPD (final concentration: 5x10⁶ cells/ml).
4. Incubate at 30°C, 200rpm until density reaches 2x10⁷ cells/ml (~4 hours).
5. Harvest cells: centrifuge at 3000g for 5 minutes.
6. Wash cells with 25ml sterile H₂O, and centrifuge again.
7. Resuspend cell pellet in 1.0ml of 0.1M LiAc, transfer to 1.5ml microfuge tube.
8. Centrifuge at top speed for 5 seconds, and remove LiAc.
9. Resuspend cells in 0.1M LiAc to a final volume of 500μl (about 400μl LiAc).
10. Prepare carrier DNA: boil 1.0ml sample for 5 minutes, then chill on ice.
11. Aliquot 50μl of cell suspension into labeled microfuge tubes, centrifuge, and remove LiAc.
12. Add transformation mix in this order:
• 240μl PEG 3350 (50% w/v)
• 36μl 1.0M LiAc
• 25μl single-stranded carrier DNA (2.0mg/ml)
• 50μl H₂O + plasmid DNA (0.1-10μg)
13. Vortex vigorously until the cell pellet is completely mixed (~1 minute).
14. Incubate 30 minutes at 30°C.
15. Heat shock for 30 minutes at 42°C.
16. Centrifuge at 6000-8000rpm for 15s, remove transformation mix.
17. Resuspend pellet in 0.2ml sterile H₂O.
18. Plate cells in different concentrations on selective plates. Recommended:
• 10μl cells + 100μl H₂O
• 200μl cells undiluted
19. Incubate plates at 30°C for 3 days.

Adapted from [29].

• Yeast culture plate
• 200mM LiOAc, 1% SDS solution
• 96% ethanol
• 70% ethanol
• H₂O

• Microcentrifuge
• Vortex mixer
• Water bath or heat block (70°C)
• Micropipettes and sterile tips
• Microcentrifuge tubes

1. Pick one yeast colony from the plate and transfer the culture to a new plate or liquid medium.
2. With the same pipette tip, suspend cells in 100 μl of 200mM LiOAc, 1% SDS solution.
3. Incubate for 5 minutes at 70°C.
4. Add 300 μl of 96% ethanol, vortex.
5. Spin down DNA and cell debris at 15,000 g for 3 minutes.
6. Wash pellet with 70% ethanol.
7. Dissolve the pellet in 100 μl of H₂O or TE and spin down cell debris for 15 seconds at 15,000 g.
8. Prepare PCR reaction (example using GoTaq™ Green Master Mix by Promega):
• GoTaq™ Green Master Mix, 2X: 12.5μl
• Forward primer, 10μM: 1μl
• Reverse primer, 10μM: 1μl
• DNA solution obtained from above: 1μl
• Nuclease-free water to 25μl

Adapted from [28].

• C. reinhardtii cells (wall-less or walled cells treated with autolysin)
• TAP medium
• Ice
• Sucrose
• Transforming DNA
• Salmon sperm carrier DNA (optional)
• Water bath
• 20% sterilized corn starch suspension
• PEG 8000
• Agar plates with TAP medium and selective agent

• Gyratory shaker
• Centrifuge
• BioRad electroporation cuvette (4 mm gap)
• BioRad Gene Pulser Xcell electroporator
• Incubator tent with light

1. Cell Culture
   • Grow cells in TAP medium at 27°C under light (50 μm photons/m² s⁻¹) on a gyratory shaker at 120 rpm until late exponential phase.
   • Chill cells on ice and harvest by centrifugation (880 × g for 5 min).
   • Resuspend cells in TAP + 40 mM sucrose at (1-4) × 10⁷ cells/mL.
2. Electroporation
   • Place 250 μL of concentrated cells at 16°C in the electroporation cuvette.
   • Add 2.5 μg transforming DNA and 50 μg salmon sperm carrier DNA (optional) to the cuvette.
   • Place cuvette in the electroporation device set at 1800-2300 nV/cm with capacitance at 10 μF and no shunt resistor.
   • Perform electroporation (time constant should be 5-6 ms).
3. Post-electroporation
   • Place the cuvette containing electroporated cells in a 25°C water bath for 5-30 min for recovery.
4. Plating
   • Prepare a suspension of 1 mL 20% sterilized corn starch in TAP + 40 mM sucrose + 0.4% PEG 8000.
   • Add an aliquot (0.4%-2% of sample) of electroporated cells to the suspension.
   • Spread the mixture onto plates containing TAP medium in 0.5% agar + agent for selective growth of transformants.
5. Incubation
   • Incubate plates under light (80 μm photons/m² s⁻¹) at 27°C until colonies are large enough for analysis.

Note: The expected transformation rate is approximately 10⁵ transformants/μg DNA.

Adapted from [6].

• Strains
  • Escherichia coli:
    • NCM 3722 prototrophic K-12 strain with PlsB-msGFP2 integrated into the chromosome
    • DH10B E. coli: (F– mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara leu) 7697 galU galK rpsL nupG λ–)
    • JW2880-1 strain: (F-Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, ΔserA764::kanR, rph-1, Δ(rhaD-rhaB)568, hsdR514)
  • Saccharomyces cerevisiae:
    • S. cerevisiae ρ+ NB97: (MATa leu2-3,112 lys2 ura3-52 his3ΔHindIII arg8Δ::URA3 [cox2-60::ARG8m])
    • S. cerevisiae ρo MTCC109: (ATCC201440)
• Growth Media
  • E. coli:
    • LB medium (no antibiotics)
    • 2YT medium
    • Minimal agar medium (M9 medium with Casamino Acids – Vitamin Assay)
  • S. cerevisiae:
    • YPD medium (1% Bacto yeast extract, 2% Bacto peptone, 2% glucose)
    • Modified YPD (with 0.1% glucose/3% glycerol or 3% glycerol, and 1 M sorbitol)
    • YPDS medium (YPD medium supplemented with 1 M sorbitol)
• Selection Media
  • Selection Medium II: 1% Bacto yeast extract, 2% Bacto peptone, 3% glycerol, 0.1% glucose, 1 M sorbitol, 1.5% agar, 1 mM arabinose
  • Selection Medium III: 1% Bacto yeast extract, 2% Bacto peptone, 3% glycerol, 1 M sorbitol, 1.5% agar, 1 mM arabinose (without glucose)
• Reagents
  • Bacterial resuspension buffer: 10 mM Tris-HCl, 2.5 mM MgCl2, 10 mM CaCl2, pH 8
  • PEG buffer: 20% PEG 8000, 10 mM Tris-HCl, 2.5 mM MgCl2, 10 mM CaCl2, pH 8
  • TSC buffer: 10 mM Tris-HCl, 10 mM CaCl2, 1 M sorbitol, pH 7.5
  • 1 M sorbitol solution
  • 4 M sorbitol solution
  • Zymolyase 100T
• Additives and Antibiotics
  • 10 µM thiamin
  • 100 µM NAD
  • 50 mg/L kanamycin
  • 50 mg/L chloramphenicol
  • 5 mg/L tetracycline
  • 1 mM arabinose

• Incubator/shaker (30°C and 37°C)
• Water bath (37°C)
• Centrifuge (capable of 1,500g, temperature control)
• Spectrophotometer (for OD600 measurements)
• Sterile culture tubes and flasks
• Microcentrifuge tubes
• Petri dishes
• Pipettes and tips
• Vortex mixer
• Refrigerator (4°C)
• Laminar flow hood or Bunsen burner (for sterile technique)

1. Preparation of E. coli Cells:
   • Grow E. coli cells (expressing GFP) in 5 mL of LB medium until OD600 ~ 0.8.
   • Harvest the cells at 4°C and wash twice with chilled bacterial resuspension buffer (10 mM Tris-HCl, 2.5 mM MgCl2, 10 mM CaCl2, pH 8).
   • Resuspend the cells in 500 µL of resuspension buffer.
2. Preparation of Yeast Spheroplasts:
   • Grow yeast cells in YPD medium overnight.
   • Harvest the cells and wash twice with sterile water, then twice with 1 M sorbitol solution (20 mL per gram of cells).
   • Resuspend the cells in sterile-filtered 1 M sorbitol solution (5 mL per gram of cells) containing Zymolyase 100T (5-10 mg/g of cells).
   • Incubate the suspension in a water bath at 37°C for 1 hour to generate spheroplasts.
   • Cool the spheroplast suspension on ice for 20-30 minutes, then centrifuge at 1,500g, 4°C for 10 minutes.
   • Wash the spheroplasts twice with 1 M sorbitol solution (5 mL per gram of cells) and resuspend in 1 M sorbitol solution (2 mL per gram of cells).
3. Fusion Procedure:
   • Mix 500 µL of spheroplast suspension with 500 µL of TSC buffer, incubate at 30°C for 10 minutes, then centrifuge at 1,500g for 10 minutes.
   • Resuspend the spheroplasts in 100 µL of TSC buffer.
   • Mix 150 µL of bacterial cell suspension quickly with 50 µL of 4 M sorbitol, then immediately add the mixture to 100 µL of spheroplast suspension in TSC buffer.
   • Invert the tubes to mix and incubate at 30°C for 10 minutes.
   • Add the mixture to 2.5 mL of PEG buffer and incubate at 30°C for 45 minutes.
   • Centrifuge at 1,500g, 25°C for 10 minutes. Discard the supernatant PEG solution.
4. Post-Fusion Processing:
   • Slowly add 1 mL of YPDS medium to the cell pellet without disturbing the pellet and incubate at 30°C for 2 hours.
   • Partially resuspend the pellet by tapping the side of the tube and incubate in a 30°C shaker at 70 rpm for 3 hours.
   • Pellet the cells by centrifugation at 1,500g at room temperature, resuspend in 1 M sorbitol, and plate on selection medium II.
   • Overlay the plated cells with the same selection medium preincubated at 50°C (1.5% agar).
   • Incubate the plates at 25°C for 3-4 days.
5. Subsequent Culturing:
   • For subsequent rounds of growth, plate the cells on selection medium II or selection medium III as indicated.

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