{% extends "layout.html" %} {% block title %}Engineering Success{% endblock %} {% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %} {% block page_content %}
Engineering

Our iGEM project aims to develop a suitable environment for the nitrogen-fixing organelle Candidatus Atelocyanobacterium thalassa (UCYN-A) or “nitroplast" in the model organisms ​Saccharomyces cerevisiae and Chlamydomonas reinhardtii. To achieve this, we have divided the project into several key engineering modules, iterating through the design-build-test-learn cycle to refine our approach and address challenges encountered along the way.

Dry-lab characterization of uTP sequences
Cycle 1

The plan for finding a uTP sequence was initially based on results from Coale et al (2024): identify the conserved C-terminal region, find repeating motifs, and create a consensus sequence.

The analysis and filtering code was implemented in Python and the MEME suite (ref) was used for motif analysis.

We visualized the motifs and their co-occurrences.

We found that there are multiple variations in which motifs occur, yielding different uTP variants, with some shared properties.

Cycle 2

We decided to test whether certain uTP variations correlate with certain sequences.

Machine learning classifiers were trained on the sequence data to predict the corresponding uTP motifs from the protein’s main body.

The classifiers were evaluated using 5-fold cross-validations and permutation test.

We found that 3 of the classifiers have indicated a statistically significant correlation between protein sequences and motif variations. We used the top classifier, Logistic Regression, to design a uTP sequence for our fluorescent proteins.

Cycle 3

To evaluate the constructed fluorescent uTP proteins, we decided to investigate their 3D structure using prediction tools.

The structures of al uTP-containing B. bigelowii proteins and the uTP constructs were predicted using AlphaFold 3.

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

Analysis identified a region in the uTP sequences exhibiting a highly conserved structure. The superimposition of this region onto our construct structures demonstrated a correspondence between the two.

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