iGEM 2024 — Team tu-delft

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{% extends "layout.html" %} {% block title %}Engineering Success{% endblock %}
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Engineering

UCYN-A transit peptide sequences

uTP Dry-lab Analysis - Cycle 1

uTP Dry-lab Analysis - Cycle 2

uTP Dry-lab Analysis - Cycle 3

Plasmid Construction - Cycle 1

Plasmid Construction using DpnI...

Saccharomyces cerevisiae Transformation

PEG Fusion - Cycle 1

PEG Fusion - Cycle 2

Chlamydomonas reinhardtii Transformation

Project Engineering

Our iGEM project focuses on creating a suitable environment for the
nitrogen-fixing organelle
*Candidatus Atelocyanobacterium thalassa* (UCYN-A), commonly
referred to as the "nitroplast," within the model organisms
*Saccharomyces cerevisiae (S. cerevisiae)* and
*Chlamydomonas reinhardtii (C. reinhardtii)*. These eukaryotic
organisms were chosen not only for their well-characterized genetic
systems and ease of manipulation but also for their relevance to our
long-term goals. Specifically, *Chlamydomonas* is a plant-like
organism, aligning with our ultimate aim of introducing the nitroplast
into crops for sustainable nitrogen fixation.

We approached the project through a series of iterative engineering
steps, utilizing the design-build-test-learn (DBTL) cycle to
systematically address challenges and refine our protocols. This
approach allowed us to continuously improve and adapt as we progressed.

Identifying and characterizing the UCYN-A transit peptide sequences

The first step on our roadmap was to identify the UCYN-A Transit
Peptide (uTP) sequence, which is essential for importing
host-encoded proteins into UCYN-A. This process is crucial because
protein import into UCYN-A must be understood and replicated to
develop a successful symbiotic relationship between the organelle
and a new eukaryotic host. By enabling the import of specific host
proteins into UCYN-A, this step lays the foundation for
transplanting UCYN-A into a model organism.

Our dry lab team used bioinformatics tools, including multiple
sequence alignment (MSA) and motif analysis, to identify conserved
motifs in uTP sequences from the proteomics data of
*Braarudosphaera bigelowii (B. bigelowii)*
[[1]](#cite1). These C-terminal extensions exhibited
characteristics similar to transit peptides in mitochondria and
chloroplasts, and we hypothesized that these sequences could direct
protein import into UCYN-A.

To investigate the in vivo behavior of the identified uTP sequences,
we engineered plasmids containing uTP fused to fluorescent proteins.
Our aim was twofold: first, to observe the localization of
uTP-marked proteins in cells lacking UCYN-A, which could provide
insights into potential relationships between the UCYN-A protein
import system and other known mechanisms. Second, expressing a
uTP-containing protein would enable us to purify it, inject it into
live B. bigelowii, and monitor its localization in the native host,
thereby confirming uTP's function as a targeting signal.

For *C. reinhardtii*, we used the plasmid backbone
pOpt2-mVenus-Ble [[6]](#cite6) encoding mVenus, a yellow
fluorescent protein, as this protein has a lower background
fluorescence in *C. reinhardtii* compared to green
fluorescent protein (GFP), as noted by [[2]](#cite2). For
*S. cerevisiae*, we chose the pUDE1311
[[7]](#cite7) plasmid encoding mNeonGreen.

Additionally, we created control plasmids containing mitochondrial
transit peptides (MTS1) [[8]](#cite8) and chloroplast
transit peptides (CTS) [[9]](#cite9). These controls
would allow us to compare the localization of uTP-tagged proteins
with those that are known to localize to specific organelles in
*S. cerevisiae* and *C. reinhardtii*. This comparison
lets us assess whether uTP shows similar or distinct localization
patterns.

During the testing phase, we attempted to transform
*S. cerevisiae* and *C. reinhardtii* with the plasmids
to observe protein localization using fluorescence microscopy.
Unfortunately, we were unable to successfully construct plasmids
with the uTP sequence incorporated. However, we managed to construct
and image plasmids containing MTS1 in *S. cerevisiae*, which
showed correct localization to the mitochondria as expected.

From this initial round of testing, we learned that while we
successfully confirmed mitochondrial localization for MTS1, the uTP
plasmid construction did not succeed.

Although we lacked results for uTP, this partial success with MTS1
suggests that our experimental pipeline works for mitochondrial
localization. The failure with the uTP constructs highlights the
need for further refinement in plasmid assembly and transformation.

As a next step, we plan to reattempt the assembly of the plasmid
with the uTP sequence and repeat the transformations. Once the uTP
plasmids are successfully constructed, we aim to test their
localization properties in eukaryotic hosts like
*S. cerevisiae* and *C. reinhardtii* to confirm
whether the uTP sequence can direct protein import into UCYN-A.

uTP Dry-lab Analysis - Cycle 1

Our initial plan for identifying the uTP sequence was based on the
results from Coale et al. (2024). We aimed to identify the conserved
C-terminal region, find repeating motifs, and create a consensus
sequence.

To implement our analysis plan, we developed Python code for
sequence analysis and filtering. For motif analysis, we utilized the
MEME suite, a powerful tool for discovering and analyzing sequence
motifs.

We visualized the identified motifs and their co-occurrences to
better understand the patterns within the uTP sequences.

Through this analysis, we discovered that there are multiple
variations in which motifs occur, resulting in different uTP
variants. Despite the variations, we observed some shared properties
among these variants.

uTP Dry-lab Analysis - Cycle 2

Based on our findings from the first cycle, we decided to
investigate whether certain uTP variations correlate with specific
protein sequences. This could provide insights into the relationship
between protein structure and uTP variation.

To test this hypothesis, we implemented machine learning classifiers
trained on the sequence data. These classifiers were designed to
predict the corresponding uTP motifs from the protein's main body.

We evaluated the performance of our classifiers using 5-fold
cross-validations and permutation tests. These rigorous testing
methods helped ensure the reliability of our results.

Our analysis revealed that three of the classifiers indicated a
statistically significant correlation between protein sequences and
motif variations. Among these, the Logistic Regression classifier
performed best. We used this top-performing classifier to design a
uTP sequence for our fluorescent proteins.

uTP Dry-lab Analysis - Cycle 3

To further evaluate the constructed fluorescent uTP proteins, we
decided to investigate their 3D structure using prediction tools.
This would allow us to compare the structural features of our
designed uTP sequences with those of native uTP-containing proteins.

We used AlphaFold 3, a state-of-the-art protein structure prediction
tool, to predict the structures of all uTP-containing
*B. bigelowii*
proteins and our uTP constructs.

The predicted structures were analyzed using PyMol and Python. We
created a consensus structure from the native uTP sequences to
highlight the structurally conserved regions.

Our analysis identified a region in the uTP sequences exhibiting a
highly conserved structure. When we superimposed this region onto
our construct structures, we observed a clear correspondence between
the two. This finding suggests that our designed uTP sequences
likely maintain the crucial structural features necessary for their
function.

Plasmid Construction - Cycle 1

In this cycle, our goal was to construct plasmids that would express
fluorescent proteins fused to uTP or control sequences in
*S. cerevisiae* and *C. reinhardtii*. To ensure proper
incorporation of the inserts, we designed primers with specific
overhangs for PCR amplification. We planned to linearize the plasmid
backbones (pOpt2-mVenusBle for *C. reinhardtii* and pUDE1311
for *S. cerevisiae*) at the C-terminal and N-terminal regions
for precise insertion of the uTP, MTS1, and CTS sequences.

The assembly of plasmids containing the uTP, MTS1, and CTS sequences
was then completed using the amplified backbones and inserts with
overlapping ends. For this purpose, we used Gibson Assembly to
combine the linearized plasmid backbones with the uTP, MTS1, and CTS
inserts due to its efficiency in assembling multiple DNA fragments
with overlapping ends. We aimed to confirm the correct plasmid
assembly by transforming *E. coli* with the resulting
constructs and performing diagnostic tests.

We amplified the plasmid backbones using KOD polymerase and
incorporated the appropriate overhangs into our PCR products. The
C-terminal and N-terminal regions of both the
*C. reinhardtii* and *S. cerevisiae* plasmids were
successfully linearized. We then carried out Gibson Assembly using
the amplified plasmid backbones and inserts.

After excising the correct fragments and assembling the plasmids
using Gibson Assembly, we ran additional diagnostic tests, including
diagnostic PCR on the Gibson Assembled plasmid. In the case of the
*C. reinhardtii* plasmid, we observed multiple fragments
during gel electrophoresis, suggesting incomplete or incorrect
assembly.

We also transformed *E. coli* cells with the assembled
plasmids to test for successful construct formation. To confirm the
successful incorporation of the inserts into the plasmids, we
conducted colony PCR on the *E. coli* colonies. For the
*C. reinhardtii* plasmid, the colony PCR yielded promising
results, indicating successful assembly, which was later invalidated
since the sequence was incorrect. However, for the
*S. cerevisiae* plasmid, many colonies showed false
positives, with only the plasmid backbone (without the insert)
present.

Through this process, we learned that, in the case of the
*C. reinhardtii* and *S. cerevisiae* N-terminal
constructs, using primers with non-specific binding can lead to the
generation of multiple fragments during gel electrophoresis, leading
to complexities in plasmid construction. We excised the fragment of
the correct size, but this approach raised concerns about potential
DNA damage from UV exposure during gel excision, which could lower
the yield or affect the quality of the DNA. For future plasmid
construction, we plan to design new primers to avoid excision
altogether and explore using restriction digestion instead of Gibson
Assembly to increase the accuracy of our insertions.

Upon closer investigation, we also discovered that the false
positives in the colony PCR of the *S. cerevisiae* constructs
were likely due to leftover circular pUDE plasmid from the backbone
amplification process. The ratio of Gibson product (with insert) to
non-linearized backbone was too similar, leading to a higher
proportion of colonies without the desired insert. This reduced the
efficiency of Gibson Assembly. To address this issue, we decided to
use DpnI digestion.

Plasmid Construction using DpnI for restriction digestion during E.
Coli transformation

In this cycle, our goal was to transform *E. coli* with the
resulting plasmid constructs and perform diagnostic tests. However,
this time, we used DpnI for restriction digestion. DpnI is a
restriction enzyme that cuts at methylated sites to eliminate any
leftover circular plasmid from the backbone amplification.

We once again amplified the plasmid backbones using KOD polymerase
and incorporated the appropriate overhangs into our PCR products.
The C-terminal and N-terminal regions of
*S. cerevisiae* plasmid were successfully linearized. We then
carried out Gibson Assembly using the amplified plasmid backbones
and inserts. Following this, we transformed E. Coli cells with the
constructed plasmids.

To confirm the successful incorporation of the inserts into the
plasmids, we conducted colony PCR on the transformed
*E. coli* colonies.

After applying DpnI digestion, the transformation efficiency
improved, and we obtained positive colonies verified by colony PCR.
From this cycle, we learned that the leftover circular plasmid from
the amplification process can significantly reduce the efficiency of
Gibson Assembly and lead to false positives during colony PCR. By
using DpnI digestion, we were able to remove the unwanted circular
plasmid and increase the transformation efficiency. Moving forward,
we plan to incorporate DpnI digestion as a standard step when using
amplified plasmid backbones to ensure that only linearized DNA is
used in the assembly.

*Saccharomyces cerevisiae* Transformation

In this cycle, we aimed to transform *S. cerevisiae* with the
plasmids successfully assembled in the previous cycle. These
plasmids contained fluorescent protein constructs (mNeonGreen)
tagged with either the mitochondrial transit peptide (MTS1) or
UCYN-A Transit Peptide (uTP2), alongside a control without transit
peptide (mNeonGreen only) and a negative control without plasmid.

To select for successfully transformed yeast cells, we used the URA3
gene as a selective marker, allowing growth in uracil-deficient
media. Our goal was to confirm successful transformation and
integration of the plasmids into *S. cerevisiae* for
subsequent localization studies.

We followed a yeast transformation protocol adapted from
[[3]](#cite3). After transforming the yeast with the
mNeonGreen-MTS1, mNeonGreen-uTP2, and control plasmids, we plated
the cells on uracil-deficient media to select for successful
transformants.

Colonies that formed on the selective plates were isolated, and
diagnostic PCR was performed on 9 colonies from the mNeonGreen-MTS1
transformation to confirm the presence of the MTS1 transit peptide
sequence. Out of the 9 colonies tested, 8 showed positive results
for the MTS1 sequence, indicating successful transformation.
However, none of the colonies transformed with the mNeonGreen-uTP2
construct showed positive results.

To test the results of the transformations, we repeated the
transformation with the uTP2 construct to rule out low
transformation efficiency as the cause of the negative results.
After the second round of transformation, the colonies formed more
slowly and did not grow as well as those transformed with the MTS1
construct.

We inoculated the cells in liquid media supplemented with the
appropriate selective antibiotics, and after 3 days, the cultures
grew successfully. Colony PCR confirmed the presence of the correct
inserts in the colonies for the MTS1 construct, but microscopy
imaging revealed no significant fluorescent signal in the cells
transformed with the uTP2 construct.

We sequenced the Gibson assembly constructs using Plasmidsaurus,
which revealed that the backbones of the uTP2 plasmid were
incorrect, explaining the lack of fluorescence. This discovery
highlighted the importance of sequencing the constructs before
proceeding to transformation. Moving forward, we plan to sequence
the plasmids immediately after assembly to avoid unnecessary
transformation steps with incorrect constructs.

PEG Fusion - Cycle 1

Our goal in this cycle was to design a protocol for fusing UCYN-A
into a eukaryotic host cell. As a proof of concept, we aimed to
replicate the fusion protocol described by [[4]](#cite4),
which uses polyethylene glycol (PEG) to make host cell membranes
more permeable, allowing for the fusion of *E. coli* into
*S. cerevisiae*. We adapted the protocol by varying the
concentrations of zymolyase (an enzyme used to digest the yeast cell
wall), and we used *E. coli* and
*S. cerevisiae* strains that were auxotrophy-independent,
meaning they did not need to rely on each other for survival.

To track the success of the fusion under the microscope, we used
*E. coli* NCM3722 expressing PlsB-msGFP2, which would allow
us to observe the GFP fluorescence inside the yeast cells if
successful fusion occurred.

We executed our adapted PEG fusion protocol by treating
*S. cerevisiae* cells with zymolyase to make the cell walls
more permeable, followed by PEG treatment to facilitate the fusion
with *E. coli* cells. The cells were then prepared for
microscopy imaging to observe the fusion results.

After completing the protocol, we imaged the samples using scanning
confocal microscopy to detect any fluorescent
*E. coli* inside *S. cerevisiae* cells. Unfortunately,
the results were inconclusive. While some fluorescence was observed,
it was not localized to the yeast cells, and no clear evidence of
*E. coli* inside *S. cerevisiae* was found.
Additionally, some yeast cells exhibited strong autofluorescence,
which interfered with the measurements.

From this initial round of testing, we concluded that several
factors may have contributed to the lack of conclusive results:

1. The strong autofluorescence in *S. cerevisiae* cells could
   have masked the GFP signal from *E. coli*.
2. The zymolyase concentration used may not have been sufficient to
   adequately digest the yeast cell wall, preventing successful
   fusion.
3. The ratio of *S. cerevisiae* to *E. coli* cells may
   not have been optimal for fusion.

In light of these findings, we planned a follow-up experiment with
control samples to determine the cause of the observed fluorescence.
We also considered the use of additional staining methods, such as
methylene blue to assess yeast cell viability and Concanavalin-A
FITC to visualize whether the yeast cell wall had been fully
digested.

PEG Fusion - Cycle 2

After the initial PEG fusion experiments yielded inconclusive
results, we designed a follow-up set of experiments to address the
key issues we identified, such as autofluorescence in
*S. cerevisiae* and insufficient zymolyase concentration. Our
objective was to test the hypothesis that the observed fluorescence
in the yeast cells might have been due to autofluorescence or
improper cell wall digestion.

To test this hypothesis, we planned control samples and introduced
additional staining methods to clarify the source of the
fluorescence:

1. *S. cerevisiae* treated with PEG but without
   *E. coli* (to control for autofluorescence).
2. *E. coli* added to *S. cerevisiae* without PEG
   treatment (to control for random bacterial association).
3. Repetition of the original PEG fusion protocol with two different
   zymolyase concentrations.
4. Use of methylene blue staining to check yeast viability (live
   cells should not take up the stain).
5. Use of Concanavalin-A FITC to visualize whether the yeast cell
   wall had been successfully digested.

We prepared the control samples and repeated the PEG fusion protocol
using the adjusted zymolyase concentrations. After the fusion
process, the cells were stained with methylene blue and
Concanavalin-A FITC to assess cell wall integrity and cell
viability.

Once the staining was complete, the samples were prepared for
imaging with confocal microscopy to evaluate fluorescence and
compare the controls to the experimental samples.

We imaged the control and experimental samples using scanning
confocal microscopy to detect GFP fluorescence and assess cell
viability. Additionally, we performed optical microscopy to check
yeast viability using methylene blue staining.

The results showed no significant difference between the control
samples and the full protocol experiments. The same fluorescence
observed in some *S. cerevisiae* cells appeared across all
conditions, confirming that the signal was likely due to
autofluorescence. Furthermore, *E. coli* was not visible in
the yeast cells, which suggested that the GFP expression was too
weak under the current conditions.

From this second cycle of testing, we learned that the
autofluorescence signal in *S. cerevisiae* cells was likely
masking the GFP signal from *E. coli*, as we observed similar
fluorescence in the control samples. Additionally, the weak GFP
expression in *E. coli* may have further contributed to the
lack of observable fusion events.

To improve the fusion protocol, we plan to:

1. Use DAPI staining, which binds to DNA, to detect
   *E. coli* without interference from yeast autofluorescence.
2. Consider using a stronger promoter to enhance GFP expression in
   *E. coli*, ensuring that any fusion events are more easily
   detectable in future experiments.

*Chlamydomonas reinhardtii* Transformation

The objective of this cycle was to transform
*Chlamydomonas reinhardtii* with the mVenus-insert fused to
different peptide sequences: the UCYN-A Transit Peptide (uTP2),
mitochondrial transit peptide (MTS1), and chloroplast transit
peptide (CTS). The goal was to test whether these peptides could
direct the localization of the fluorescent proteins to their
respective compartments within the *Chlamydomonas* cells.

We first designed a transformation protocol based on the T-sorbonne
*Chlamydomonas* guide and a transformation protocol by
[[5]](#cite5). The UVM4 strain of
*C. reinhardtii*, obtained from the Planck Institute, was
chosen for its high transformation efficiency. For the
transformation, we selected the pOpt2-mVenus-Ble plasmid backbone,
which contains specific introns in the mVenus coding sequence to
prevent gene silencing in *Chlamydomonas* and strong
promoters to drive expression.

We prepared the mVenus-insert by performing restriction digestion
around the mVenus-insert fragment, followed by SV gel and PCR
cleanup to isolate the insert. Due to inefficient cleanup methods
that resulted in a low yield of the insert, we repeated the
digestion and switched to ethanol precipitation, which improved the
recovery of the insert for transformation.

Electroporation was performed using the protocol we designed, and
the transformed cells were plated on zeocin-resistant agar plates.
In the first transformation attempt, we used too much liquid and a
high cell density, which left the plates too wet and caused poor
colony formation. For the second attempt, we optimized the
conditions by reducing the liquid volume and cell density, resulting
in clearer plates with distinct colonies.

Following the second transformation and plating attempt, the
optimized conditions led to the formation of distinct
zeocin-resistant colonies. We selected 12 colonies for each
construct (mVenus-CTS, mVenus-MTS1, mVenus-uTP2, mVenus) and
screened them for fluorescence in a 96-well plate format to test
whether the transformed colonies expressed mVenus with the peptide
sequences attached.

From the initial screening results, we observed that only one colony
transformed with the mVenus control showed fluorescence. The absence
of fluorescence in the other constructs suggests that the
transformation efficiency or expression of the peptide-fused
fluorescent proteins may need further

In future experiments, we would focus on improving the expression of
the peptide-tagged mVenus proteins. Specifically, verifying the
quality and yield of the insert before transformation would help
ensure successful expression in *C. reinhardtii*.

References

1. Coale, T. H., et al. (2024). Nitrogen-fixing organelle in a marine
   alga. Science, 384(6692), 217–222.
   <https://doi.org/10.1126/science.adk1075>
2. Neupert, J., Karcher, D., & Bock, R. (2008). Generation of
   Chlamydomonas strains that efficiently express nuclear transgenes.
   The Plant Journal, 57(6), 1140–1150.
   <https://doi.org/10.1111/j.1365-313x.2008.03746.x>
3. Susan, W., & Jean-Marc, D. (2014). P.24 High Efficiency
   Transformation of Yeast. Industrial Microbiology, Delft University
   of Technology.
4. Mehta, A., Supekova, L., Supek, F., & Schultz, P. (2018).
   Engineering yeast endosymbionts as a first step towards laboratory
   evolution of mitochondria. The FASEB Journal, 32(S1).
   <https://doi.org/10.1096/fasebj.2018.32.1_supplement.530.3>
5. Weeks, D. P. (2023). Genetic transformation of Chlamydomonas
   nuclear, chloroplast, and mitochondrial genomes. In Elsevier eBooks
   (pp. 325–343).
   <https://doi.org/10.1016/b978-0-12-822457-1.00018-2>
6. iGEM Kaiserslautern & iGEM Sorbonne Universite. (2019). The Chlamy
   Guide.
7. 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>
8. 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>
9. 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>
10. Langenberg, L. (2023). Part:BBa K4806014 - parts.igem.org. Igem.org.
    [https://parts.igem.org/Part:BBa\_K4806014](https://parts.igem.org/Part%3ABBa_K4806014)

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Materials and Methods

UCYN-A Transit Peptide Sequence

*B. bigelowii* Proteome Analysis

Plasmid Design

*E. coli*

*S.cerevisiae*

*C. reinhardtii*

PEG Fusion

Data and Code

Lab Notebook

Protocols

References

UCYN-A Transit Peptide Sequence

UCYN-A enriched proteins were selected from a protein quantification dataset [[26]](#cite26). Multiple sequence alignment was performed on these proteins using COBALT [[15]](#cite15) with default parameters, followed by alignment cleanup using CIAlign [[16]](#cite16) 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]](#cite18) 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]](#cite19) model, obtained from HuggingFace Model Hub. Logistic regression, decision tree, random forest, and support vector machine classifiers, as implemented by scikit-learn [[20]](#cite20) 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]](#cite21), 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]](#cite22) 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]](#cite23). 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]](#cite25). 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.

*B. bigelowii* Proteome Analysis

Using raw RNA-seq data from [[27]](#cite27), a transcriptome assembly was constructed using DRAP [[35]](#cite35) with the oases assembler and default parameters. The completeness of the assembly was tested with BUSCO [[33]](#cite33). Proteins were predicted from the transcriptome assembly with TransDecoder [[34]](#cite34). Ortholog analysis was performed using OrthoFinder [[36]](#cite36). See [Data and Code](#eight) for the predicted proteome.

Plasmid Design and Construction

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]](#cite13), mTP (mitochondrial transit peptide)[[38]](#cite38) and cTP (chloroplast transit peptide) [[14]](#cite14). 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]](#cite11), 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]](#cite9) [[10]](#cite10), 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]](#cite1), following the manufacturer’s protocol [[2]](#cite2). pUC19 and a DNA fragment provided by the kit were used as positive controls.

See [Parts and Sequences](/tu-delft/parts/) for more details on the design and sequence of our parts.

*Escherichia coli* Transformation and Verification

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]](#cite3).

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.

*Saccharomyces cerevisiae* Transformation

*S. cerevisiae*  CEN.PK113-5D (full genome available at [[12]](#cite12)), 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.

*Chlamydomonas reinhardtii* Transformation

The UVM4 strain of *C. reinhardtii* [[31]](#cite31) 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]](#cite9). 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]](#cite30) and [[37]](#cite37) 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.

Polyethylene Glycol (PEG) Fusion

A protocol for fusing *E. coli*  into *S. cerevisiae*  using polyethylene glycol (PEG) was adapted from [[6]](#cite6). 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.

Data and Code

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](https://gitlab.igem.org/mablin/utp-sequence-analysis).

The list of selected *B. bigelowii* protein sequences with C-terminal uTP can be downloaded [here](%7B%7B%20url_for%28%27static%27%2C%20filename%3D%27good-c-term-full.fasta%27%29%20%7D%7D)

The consensus structure obrained for uTP can be downloaded [here](%7B%7B%20url_for%28%27static%27%2C%20filename%3D%27consensus-c-term.pdb%27%29%20%7D%7D)

Our predicted *B. bigelowii* proteome is available upon request.

The sequences of all primers used in our experiments can be found here [here](%7B%7B%20url_for%28%27static%27%2C%20filename%3D%27primers.csv%27%29%20%7D%7D)

See [Parts and Sequences](/tu-delft/parts/) for more details on the design and sequence of our parts.

Lab Notebook

The lab notebook can be viewed [here](https://static.igem.wiki/teams/5054/notebook-compressed.pdf).

Protocols

Yeast Transformation

Adapted from [[7]](#cite7) following [[8]](#cite8).

Materials and Reagents

* 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

Equipment

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

Procedure

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.

Yeast Colony PCR

Adapted from [[29]](#cite29).

Materials and Reagents

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

Equipment

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

Procedure

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

*C. reinhardtii* Transformation

Adapted from [[28]](#cite28).

Materials and Reagents

* *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

Equipment

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

Procedure

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.

Yeast-*E. coli* Fusion

Adapted from [[6]](#cite6).

Materials and Reagents

* **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

Equipment

* 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)

Procedure

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.

References

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9. 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>
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{% endblock %}

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{% block title %}Results{% endblock %}
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Results

**DRY LAB**

The uTP sequence

**WET LAB**

uTP expression

UCYN-A fusion

**References**

To lay the foundation for a new avenue of research into engineering nitrogen-fixing endosymbionts, we investigated protein transport to *B. bigelowii* ’s symbiotic partner UCYN-A (nitroplast) and implemented proof-of-principle endosymbiosis experiments.

**DRY LAB**

The majority of proteins within mitochondria and chloroplasts are nuclear-encoded – they are expressed by the host and are imported into the organelle. Proteins meant for the organelle are usually marked by a targeting sequence at one end, also known as a transit peptide, which directs the protein to its destination after which it is cleaved.

This is no different with UCYN-A: Coale et al [[1]](#cite1) in their 2024 study used proteomics to find proteins encoded by the host and imported into the nitroplast. Upon examining these protein sequences, they noticed that many of them possess characteristics of organellar import – most of them possess a C-terminal 120 amino acid extension compared to their orthologues. This extension is reminiscent of targeting sequences known to exist in mitochondrial [[2]](#cite2) and chloroplastic [[3]](#cite3) imported proteins. They termed the putative targeting sequence uTP (UCYN-A Transit Peptide, with lowercase “u” to differentiate it from uridine triphosphate).

Our investigations began with an in-depth computational analysis of *B. bigelowii* ’s proteome [[1]](#cite1), [[7]](#cite7) to identify potential signals marking proteins for import into UCYN-A. Based on these results, we designed fluorescent protein-transit peptide constructs for expression in model organisms to show that the identified signals indeed localize to UCYN-A. To pave the way for transplanting the nitroplast into new organisms, we also explored the feasibility of physically inserting UCYN-A into a new host by attempting cell fusion experiments. Furthermore, we successfully established a culture of *B. bigelowii* and tested a new protocol for isolating UCYN-A. These experiments collectively aim to elucidate the mechanisms of UCYN-A's endosymbiotic relationship and lay the groundwork for future engineering of nitrogen-fixing symbionts into new host organisms.

![Fig 1: Graphical overview of the experiment plan.](https://static.igem.wiki/teams/5054/graphical-abstract.png)

Fig 1: Graphical overview of the experiment plan.

The UCYN-A transit peptide sequence

If, like other organelles, UCYN-A relies on proteins imported from the host for normal functioning, characterizing the import system and the targeting sequence is essential before transplanting the organelle into a new host organism. Building upon the work of Coale et al. [[1]](#cite1), we aimed to advance the understanding of uTP by identifying its precise sequence.

Starting from the raw proteomics data from [[1]](#cite1), we selected 368 proteins expressed by the host and significantly enriched in UCYN-A and performed multiple sequence alignment (MSA). Using the alignment we identified a strongly conserved C-terminal region in many of the imported proteins similar to that reported by [[1]](#cite1). We selected a subset of 206 proteins with highly similar (>60% sequence identity) C-terminal alignments, indicating that these are likely to contain uTP.

![Fig 1: Graphical overview of the experiment plan.](https://static.igem.wiki/teams/5054/msa.png)

Fig 2: Multiple sequence alignment (MSA) of all UCYN-A enriched sequences. The strongly aligned C-terminal region is highlighted (positions 880-1010 in the alignment)

Motif analysis confirmed findings similar to [[1]](#cite1), revealing 8 conserved motifs in the C-terminal region (Fig 2). Further investigation of motif co-occurrence and relative positioning uncovered common patterns: two motifs consistently appeared near the start of the C-terminal region at fixed positions, followed by various combinations of the remaining motifs. This arrangement is reminiscent of a potential sub-organellar localization mechanism, where the initial two motifs could target UCYN-A, while subsequent motifs may specify localization within the endosymbiont, as is the case with chloroplast targeting, where a bipartite N-terminal targeting sequence specifies stromal and thylakoidal localization. More research is needed however to investigate this hypothesis.

![Fig 1: Graphical overview of the experiment plan.](https://static.igem.wiki/teams/5054/motifs2.svg)

Figure 3: 5 uTP variations discovered among UCYN-A imported sequences. The left panel shows the relative position of conserved motifs in the different uTP variations, relative to motif #1, which was present in all examined sequences.

We investigated the relationship between transit peptide (uTP) sequences and the functional core of proteins, known as the mature domain. The mature domain is the part of a protein that remains after the transit peptide is cleaved off and performs the protein's primary function. Given the observed diversity in uTP sequences, understanding their connection to specific mature domains is crucial for designing effective uTP constructs for future experiments. Certain uTP sequences may only be compatible with specific proteins, so to explore potential correlations between uTP motif patterns and mature domain sequences, we trained classifiers to predict the appropriate uTP sequence (by predicting the correct combination of motifs) based on a given mature domain sequence. The classifiers were evaluated using a permutation test [[10]](#cite10), with 3 of them yielding statistically significant results (p < 0.05) (Fig 4).

|  | Accuracy | Permutation p-value | F1 Score |
| --- | --- | --- | --- |
| Logistic Regression | 68% | 0.0010 | 0.61 |
| Random Forest | 68% | 0.0010 | 0.49 |
| SVC | 45% | 0.0010 | 0.34 |
| Decision Tree | 9% | 0.0380 | 0.13 |

Figure 4: Classification results of 4 different classifiers trained to predict uTP sequences based on mature domains.

For in vivo characterization, we constructed candidate uTP sequences by concatenating the consensus sequences of discovered motif patterns. The uTP sequence classifiers were used to select the correct motifs for the fluorescent proteins we planned to use, mVenus and mNeonGreen. The two sequences with the highest confidence values (uTP1 and uTP2) were selected for in vivo experiments. These sequences were also submitted to the Parts Registry.

uTP1 Amino Acid Sequence

```
WLEEWRERLECWWGPVGTQTQLGACMGALGLHLGSRLDNEQETQTISAIVAEPGCEWVEEAAPGLPDFPEPFSLPPIPRL
```

uTP2 Amino Acid Sequence

```
WLEEWRERLECWWLDPKTQTQLGACMGALGLHLGSRLDIAPYFAWRAALLGRAPPPSARAEPGCEWVEEALDDLPDFPEPFSLPPIPRL
```

To further validate the constructed sequences, their predicted structure was examined. Structural prediction was performed on all 206 selected uTP-containing *B. bigelowii* proteins, to uncover the 3D conformation of uTP. The predicted structures were aligned and a consensus structure was created by averaging the aligned regions. This revealed a highly-conserved (stdev per residue position < 1.8Å) structural region with 2 alpha-helices arranged into a U-bend (Fig 5). The structure of constructed mNeonGreen and mVenus + uTP1, uTP2 sequences was predicted and the consensus structure aligned onto them, yielding good alignment (RMSD<=4.0Å), confirming that our constructs will likely behave similar to native uTP-containing proteins.

![Fig 1: Graphical overview of the experiment plan.](https://static.igem.wiki/teams/5054/structures2.png)

Figure 5: Structural predictions. (a) Consensus structure of all uTP sequences extracted from UCYN-A imported proteins. (b) Consensus structure of all uTP sequences with charged residues shown (red=negative, blue=positive). (c) Consensus structure aligned onto uTP1 + mNeonGreen construct (RMSD=4.00Å). (d) Consensus structure aligned onto uTP2 + mNeonGreen construct (RMSD=3.77Å).

In the case of mitochondrial and chloroplast imported proteins, there is a well-known overlap: numerous proteins have twin transit peptides or ambiguous targeting sequences targeting both organelles. To investigate whether there is a similar overlap between the potential UCYN-A import system and other known cellular transport systems, we used established protein localization prediction tools on the potential list of UCYN-A imported proteins. These predictions proved to be inconclusive. A large minority (28%) of them were classified as secreted (Fig 6). This suggests that the UCYN-A import system, similar to other protein transport mechanisms, might be related to the Sec system.

We also investigated both sequence and structural homologs of uTP in public databases (NCBI, PDB, AlphaFold/Proteome) and found no significant matches.

![Fig 6: Average predicted confidence for different localization categories.](https://static.igem.wiki/teams/5054/localization-classification.svg)

Figure 6: Average predicted confidence for different localization categories.

The inconclusive homology search and localization prediction results, together with the fact that in most cases transport signals are found on the N-terminal of proteins [[2]](#cite2),[[3]](#cite3),[[8]](#cite8) as opposed to the C-terminal in the case of uTP, suggest the protein import machinery associated with uTP is quite distinct from other known systems in the cell.

![Fig 1: Figure 7:  Overview of hypothesized UCYN-A protein import.](https://static.igem.wiki/teams/5054/import-overview.svg)

Figure 7: Overview of hypothesized UCYN-A protein import.

**WET LAB**

*in vivo* uTP characterization

**Expression of uTP tagged by Fluorescent proteins**

The ultimate goal with the uTP sequences we identified is to understand and confirm whether they are indeed responsible for protein import into UCYN-A. Conventional methods to check this would require a toolbox for genetic manipulation of *B. bigelowii*, not yet available and beyond the scope of this project. We therefore opted for using 2 model eukaryotes for further research on uTP’s behavior, namely *C. reinhardtii* and *S. cerevisiae*, and designed an experiment to confirm uTP’s function without modifying *B. bigelowii*.

We worked off of a *S. cerevisiae* and a *C. reinhardtii* backbone, pUDE1311 and pOpt2-mVenusBle respectively, in order to design constructs expressing
fluorescent proteins (FP) tagged by known transit peptides as well as uTP. Unmodified, pUDE1311 expresses ymNeongreen and pOpt2-mVenusBle expresses mVenus, a YFP analogue; Both express AmpR for selection on *E.coli*, and while pOpt expresses a Zeocin resistance gene for selection on *C. reinhardtii* pUDE expresses URA3 for auxotrophic selection on *S. cerevisiae* CEN.PK 113-5D, an strain with uracil knockout. We designed 2 constructs for expression in our yeast and 3 in our algae. For our yeast, one construct had uTP inserted in the C-terminus of ymNeongreen and the other had MTS1, a mitochondrial transit peptide [[2]](#cite2), inserted in the N-terminus of the fluorescent protein.
For our algae, one construct had uTP inserted in the C-terminus of mVenus, while the two others had a chloroplastic (cTP, [[3]](#cite3)) and a mitochondrial transit peptide (mTP, [[11]](#cite11)) respectively, both inserted in the N-terminus of mVenus. Plasmid maps for our constructs and vectors can be found in our Materials and Methods page.
We planned to observe the localization of uTP in the absence of UCYN-A in these species, hypothesizing based on the dry-lab analysis detailed above that we would observe uniform diffusion in the cytoplasm. Cells transformed with the regular pUDE and pOpt plasmids as well as the known transit peptides would serve as controls showing both uniform diffusion as well as localization to organelles respectively.

![Fig 1: Graphical overview of the experiment plan.](https://static.igem.wiki/teams/5054/experiment-abstract.jpeg)

Figure 8: Overview of experiment

We used Gibson assembly to construct the uTP-FP and transit-peptide-FP plasmids and transformed *E. coli* with them. The *E. coli* was plated in selective medium and each plate was screened in colony PCRs to confirm
the presence of each insert, after which positive colonies were cultured in liquid to amplify the plasmid for later isolation. With isolated plasmid constructs, both *S. cerevisiae* and *C. reinhardtii* were transformed and selected after colony PCRs for analysis.
For our transformation, selection and culturing protocols, see our [Materials and Methods](experiments) page.

![Fig 1: Graphical overview of the experiment plan.](https://static.igem.wiki/teams/5054/diagnosis.jpg)

Figure 9: Example of a diagnostic colony PCR gel: Here, we are checking for the presence of the uTP sequence on *E. coli* colonies that have grown on a selective plate. Note the false positive colonies - we hypothesize these grew after being transformed with leftover pUDE1311 used as a template to linearize for Gibson assembly.

All of our inserts appeared proper on the diagnostic PCRs. Due to technical difficulties, we were not able to send over our assembled constructs for sequencing until very late in our project - only when our sequencing results came back we noticed that our pUDE1311-uTP2 construct was in fact not
what we thought. Through some strange artifact in our Gibson assembly procedure, the uTP insert assembled into pUC19, a plasmid used as a control for the assembly. We do not know how this happened, as there was no overlap with the overhangs on the uTP insert anywhere on pUC19. In addition, a few hundred basepairs between pUC19 and the insert belong to neither, which is also puzzling. The sequencing results can be found at the bottom of our [Materials and Methods](experiments) page.
This unfortunate turn of events so late on our timeline prevented us from progressing further with our experiments, as pUC19 lacks both the fluorescent protein and a yeast promoter. However, imaging our two controls in *S. cerevisiae* transformed with pUDE1311 and our pUDE1311-MTS1 construct proved the experimental pipeline works, despite the problems with the uTP construct.

![Fig 1: Graphical overview of the experiment plan.](https://static.igem.wiki/teams/5054/mts1.png)

Figure 10: MTS1-mNeongreen yeast transformants under fluorescent confocal microscope at 1000x magnification. The clustering of fluorescence is visible and visually very similar to mitochondrial localization reported by [[6]](#cite6).

Due to time constraints coupled with a long culture time for selection after transformation, we were unable to image our *C. reinhardtii* transformants.

**Protein construct delivery into *B. bigelowii***

To verify the function of uTP in vivo, we aimed to deliver purified His-tagged uTP-mNeongreen from our transformed *S. cerevisiae* cultures into *B. bigelowii* cells via electroporation. This would be followed up by high resolution imaging to confirm localization of the fluorescent protein to the nitroplast. However, this experiment was prematurely terminated due to the failure of correct uTP assembly into the yeast backbone, as previously described.

UCYN-A isolation and fusion

***B. bigelowii* culture and UCYN-A isolation**

Thanks to the generous help of Dr. Kyoko Hagino, a pioneer in research into *B. bigelowii*, we obtained a culture of *B. bigelowii* FR-21 [[1]](#cite1). This species is known to be difficult to work with, however, we were able to find the optimal conditions and grow it in our lab in Delft, establishing, to our knowledge, the first *B. bigelowii* culture in Europe. We followed Kyoko’s advice when deciding on our culture conditions, which can be found in our [Materials and Methods](experiments) page.

![Fig 1: Graphical overview of the experiment plan.](https://static.igem.wiki/teams/5054/bbigelowii.png)

Figure 11: *Braarudosphaera bigelowii*, imaged at 1000X magnification on xenic culture medium.

To allow the close study of UCYN-A by future iGEM teams and lay some groundwork for UCYN-A transplantation efforts, we developed a new, easier protocol for isolating it from a culture of B. bigelowii, compared to the known method reported by [[1]](#cite1). While Coale et al relied on a multistep procedure with a Percoll gradient and centrifugation, we used a sorting flow cytometer after lysing of the host cells. We confirmed UCYN-A’s presence in the isolate with PCR, with primers targeting part of the 16S rRNA gene exclusive to prokaryotes.

![Fig 1: Graphical overview of the experiment plan.](https://static.igem.wiki/teams/5054/cytometry-2.png)

Figure 12: Flow cytometry plot and PCR result for the three populations we identified on *B. bigelowii* lysate.
(**a**) shows the unlysed sample of *B. bigelowii*, (**b**) shows the lysed sample with the 3 populations marked for sorting, and (**c**) shows the PCR result for the 3 populations. The third population was hypothesized to contain the highest concentration of UCYN-A.

The isolated populations are not completely pure, as each of the three fractions contained UCYN-A. However, after comparing our cytometry plots with those from [[1]](#cite1), we hypothesize the third fraction to contain the densest sample of UCYN-A. This is supported by the relative intensity of the bands in the PCR gel, with the third lane being the strongest. This suggests the third population had the highest concentration of UCYN-A, since an equal amount of cells were collected in each sample and all PCR conditions were identical, meaning the intensity is approximately proportional to the amount of DNA in the starting sample.

For future investigation we would recommend the usage of qPCR in order to more precisely quantify the presence of UCYN-A DNA in different isolates.

**PEG fusion**

After identifying the proteins that must be supplied by a host to UCYN-A and the mechanism of their import, the next step to successfully transplanting it into a new host would be the actual insertion of UCYN-A into the host cells. We investigated a protocol to execute this insertion step using *S. cerevisiae* as a host and *E. coli* as a stand-in for UCYN-A.

The successful fusion of *E. coli* into *S. cerevisiae* has been shown in literature before using polyethylene glycol (PEG) to make the host cells permeable [[5]](#cite5). As a first step we attempted to replicate these results. We used *E. coli* NCM3722 expressing PlsB-msGFP2 combined with fluorescent microscopy to validate the outcome of fusion.

![Fig 1: Graphical overview of the experiment plan.](https://static.igem.wiki/teams/5054/peg.png)

Figure 13: Scanning confocal images of a sample PEG fusion procedure (left) as well as a negative control containing regular yeast cells (right)

Despite multiple attempts at the fusion procedure, we were unable to obtain conclusive results. The yeast cells' native autofluorescence interfered with measurements using the fluorescent microscope, since GFP expression in our *E. coli* strain was not very strong: we hypothesize this is due to the stress experienced by the cells during the fusion procedure. Since the intensity from autofluorescence was too similar to the *E. coli*'s, we could not draw conclusions from our images.
To circumvent these problems, selective staining of bacterial RNA or DAPI staining of *E. coli* prior to fusion were both proposed. Due to time constraints, we could not execute any of these changes.

References

1. Coale, T. H., Loconte, V., Turk-Kubo, K. A., Vanslembrouck, B., Mak, W. K. E., Cheung, S., Ekman, A., Chen, J., Hagino, K., Takano, Y., Nishimura, T., Adachi, M., Gros, M. L., Larabell, C., & Zehr, J. P. (2024a). Nitrogen-fixing organelle in a marine alga. Science, 384(6692), 217–222.<https://doi.org/10.1126/science.adk1075>
2. 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>
3. [https://parts.igem.org/Part:BBa\_K4806014](https://parts.igem.org/Part%3ABBa_K4806014)
4. Chatzi, K. E., Sardis, M. F., Tsirigotaki, A., Koukaki, M., Šoštarić, N., Konijnenberg, A., Sobott, F., Kalodimos, C. G., Karamanou, S., & Economou, A. (2017). Preprotein mature domains contain translocase targeting signals that are essential for secretion. Journal of Cell Biology, 216(5), 1357–1369. <https://doi.org/10.1083/JCB.201609022>
5. Mehta, A. P., Supekova, L., Chen, J., Pestonjamasp, K., Webster, P., Ko, Y., Henderson, S. C., McDermott, G., Supek, F., & Schultz, P. G. (2018). Engineering yeast endosymbionts as a step toward the evolution of mitochondria. Proceedings of the National Academy of Sciences, 115(46), 11796–11801. <https://doi.org/10.1073/pnas.1813143115>
6. Kunová, N., Ondrovičová, G., Bauer, J. A., Bellová, J., Ambro, Ľ., Martináková, L., Kotrasová, V., Kutejová, E., & Pevala, V. (2017). The role of Lon-mediated proteolysis in the dynamics of mitochondrial nucleic acid-protein complexes. Scientific Reports, 7(1). <https://doi.org/10.1038/s41598-017-00632-8>
7. Suzuki, S., Kawachi, M., Tsukakoshi, C., Nakamura, A., Hagino, K., Inouye, I., & Ishida, K. (2021b). Unstable Relationship Between *Braarudosphaera bigelowii* (= Chrysochromulina parkeae) and Its Nitrogen-Fixing Endosymbiont. Frontiers in Plant Science, 12. <https://doi.org/10.3389/fpls.2021.749895>
8. Albiniak, A. M., Baglieri, J., & Robinson, C. (2012). Targeting of lumenal proteins across the thylakoid membrane. Journal of Experimental Botany, 63(4), 1689–1698. <https://doi.org/10.1093/JXB/ERR444>
9. Peeters, N., & Small, I. (2001). Dual targeting to mitochondria and chloroplasts. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1541(1–2), 54–63. [https://doi.org/10.1016/S0167-4889(01)00146-X](https://doi.org/10.1016/S0167-4889%2801%2900146-X)
10. Ojala and Garriga. [Permutation Tests for Studying Classifier Performance.](https://www.jmlr.org/papers/volume11/ojala10a/ojala10a.pdf) J. Mach. Learn. Res. 2010
11. [https://parts.igem.org/Part:BBa\_K4806011](https://parts.igem.org/Part%3ABBa_K4806011)

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