Parts

seQUESTer — Comprehensive Parts Documentation

This page documents all biological parts used in our project, organized by function. These include entry vectors for molecular cloning following the MoClo-YTK system, detection mechanism components for lead sensing, and memory system elements for recording exposure events in our yeast-based biosensor.

All parts are designed and characterized to work within the Saccharomyces cerevisiae chassis, enabling sensitive detection and effective sequestration of toxic heavy metals from contaminated water sources.

The following entry vectors are used in various assemblies of transcriptional units, as outlined by the MoClo-YTK (Kit #1000000061) system. Each vector contains specific connector parts that enable modular assembly of genetic circuits.

These parts form the core detection system for lead ions, combining constitutive promoters, lead-binding aptamers integrated into artificial riboswitches, and fluorescent reporters for signal quantification.

The memory system components enable permanent recording of lead exposure events through serine integrase-mediated DNA recombination, providing a stable biological record of contamination detection.

Composite Part Background

Our designed genetic circuit can be divided into three distinct mechanisms, each requiring individual testing for validation and confirmation of proper function. The memory mechanism of the genetic circuit is crucial for ensuring the heritable and stable expression of downstream genes that increase the tolerance and sequestration capabilities of Saccharomyces cerevisiae to lead (Pb²⁺). Based on the work of Essington et al., we have begun designing a serine integrase-dependent memory system that is viable in S. cerevisiae¹.

An important consideration when designing a serine integrase-dependent mechanism is the difference between eukaryotic and prokaryotic hosts in which the system will be expressed. In our case, two major variables were the toxicity and efficiency of the selected integrase. Through the analysis of research conducted by Xu et al., the selected serine integrase was ϕBT1².

To ensure the integrase's functionality and test its efficiency within S. cerevisiae, we designed a composite part (BBa_25JX4X9M) comprising a promoter (BBa_K2950012), coding sequence (BBa_251SUYWX), and reporter (BBa_25VD3T7D).

Serine Integrase Background

Serine integrase may cause the rearrangement of DNA, which is determined by orientation-dependent recombination sites: attP--attB and attL--attR sites³. A DNA segment is inverted when attP and attB flank it in opposing directions, producing attL and attR sites with distinct directionality. When recombination directionality factors (RDFs) are present, inversion may be reversible³. The DNA segment is excised when attL and attR are present with an RDF, leaving the corresponding site on the circular excised DNA and an attP or attB site on the chromosome³.

Our project plans to exploits the inversion functionality of serine integrase to ensure heritable and stable expression of downstream genes, only in the presence of Pb²⁺.

Design of Part

As mentioned previously our composite part is constructed using three basic parts: a promoter (pTEF2) (BBa_25JX4X9M), the coding sequence of φBT1 serine integrase (BBa_251SUYWX) and, a reverse LacZ gene flanked by φBT1 attP and attB sites (BBa_25VD3T7D).

The objective of creating the part: Test the viability of using φBT1 as the selected integrase for usage as the memory system in the final genetic circuit.

Experiments and Testing

Figure 1. Illustration of the molecular biology behind the expression of LacZ in the ONPG assay through the function of integrase.

In order to test the functionality and effectiveness of the selected φBT1 serine integrase, S. cerevisiae BY4717 was transformed with the composite part construct (BBa_25JX4X9M), a positive control (pLacZPositive), a plasmid negative control (pLacZNegative), and a yeast negative control (containing no vector transformations). After a total of three days of growth, OD₆₀₀ measurements were taken for all cultures, followed by a β-galactosidase assay using ONPG (Figure 1). The results indicate that the selected serine integrase is functional in S. cerevisiae. However, its activity and efficiency relative to the positive control are drastically lower (Figure 2). Please see Results and Experiments for more information.

Figure 2. ONPG assay results of Saccharomyces cerevisiae, normalized to cell density (OD600). Yeast with pLacZNegative show β-galactosidase activity similar to wild-type. Yeast with pIntegraseTest display significantly higher β-galactosidase activity than wild-type (Student's t-test), though lower than the positive control (pLacZPositive). Significant differences compared to yeast without an entry vector are indicated by asterisks (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Conclusions & Learning

From the results presented above, the part we have constructed (BBa_25JX4X9M) functions in S. cerevisiae BY4717 to invert a DNA sequence flanked by its specific attP and attB sites. Although the integrase is functional, its efficiency is quite low.

Moving forward, we aim to determine whether the low efficiency is due to:

• Toxicity -- Is too much integrase being expressed, leading to cell death?
• Time -- Does the integrase require more or less time to successfully invert the DNA sequence?
• Activity -- Is this the natural activity of φBT1 serine integrase?

For more information, please see Future Directions.

References