Guelph iGEM

Future Directions

Plasmid Preparation and Confirmation Overview

Plasmid Preparation and Confirmation

iGEM Guelph has created an accessible library of optimized molecular biology techniques used in plasmid preparation for MoClo YTK (John Deuber).
The next step to completing this repository is developing a protocol for BsmBI mediated golden gate assembly of multigene plasmids from pre-assembled transcriptional units. Figure 1: Golden Gate Assembly of 3 transcriptional units to form a final multi-gene plasmid, using BsmBI

Figure 1: Golden Gate Assembly of 3 transcriptional units to form a final multi-gene plasmid, using BsmBI


Once complete, any team will be able to utilize iGEM Guelph's protocols to assemble basic parts from the Deuber Lab into entry vectors (EVs), add custom genes to EVs to make unique transcriptional units (TUs), and ligate TUs together into multigene plasmids. Distributing these versatile modular cloning protocols will help advance the synthetic biology community.

Steps to Optimize BsmBI Assembly

  1. Run GGA with variations in:
    • Digestion and ligation temperatures
    • Digestion and ligation times
    • Number of cycles
    • Concentrations of reagents
  2. Comparing efficacy of colony PCR versus restriction enzyme digestion to expedite screening for successful transformants

Detection Testing Overview

Detection Testing

Time constraints and limited access to equipment prevented iGEM Guelph from characterizing the detection component of seQUESTer. As a next step, the 96-well plate fluorescence assay detailed in experiments will be conducted to gather preliminary data. Below are some considerations that will be taken.

Time of Assay

GFP requires time to be transcribed, translated, and modified before it fluoresces. Substitution of the AUG start codon for a non-cognate start codon and stress exerted by growth in lead-media may cause a delay in the expression of mature GFP. Therefore, this assay will be conducted over longer periods of time with fluorescence readings at constant intervals.

Concentrations of Lead Used

Expression of GFP depends on the probability of lead binding to the aptamer to initiate translation. This module will be tested in multiple concentrations of lead to determine what the lower limits are for detection. This data will allow us to accurately compare our system to commercially available solutions.

Density of Cells

Optical density of yeast will be normalized prior to inoculation of the 96-well plate. Various densities of yeast should be tested to account for weak fluorescent signal. A high concentration of cells may be required for visible signal.

Uptake of Lead

Yeast has a selectively permeable plasma membrane which may prevent the passive diffusion of lead into the cell. The aptamer should be tested in tandem with a transporter protein if no fluorescence is observed in the 96-well plate assay.

Cell Death

After growth in lead media, cells should be observed for cytopathic effects, stunted growth, or lysis.

Memory Testing Overview

Memory Testing

The memory component of seQUESTer is composed of 2 main parts: ΦBT1 integrase and a state switchable promoter (SSP). As of 2025, only ΦBT1 integrase activity has been assayed. Before its incorporation into our final project, both integrase activity and SSP function will be optimized.

Integrase Function

ΦBT1 was proven to have an efficiency of 20% in a colorimetric ONPG assay. See our results page for more information. Future testing of integrase will use a longer period and standardized cell density. Once more data is acquired, we can test hypotheses including:

Toxicity

Is the expression of ΦBT1 serine integrase causing reduced growth or cell viability, leading to lower expression of the reporter gene?
Culturing yeast containing ΦBT1 serine integrase against wild-type BY4741 over a longer time span will reveal if ΦBT1-containing cells have stunted growth.

Time

Does ΦBT1 serine integrase require more time to facilitate the inversion of the reporter gene?
Integrase must be transcribed, translated, and modified before it can invert modules flanked by attB and attP sites. Our ONPG assay was conducted on yeast grown over 72 hours (about 6 days). In the future, this assay should be repeated over more time to allow for integrase to act upon its target sites. This would be reflected as an exponential increase in hydrolyzed (yellow) ONPG, where our test plasmid would approach the positive control absorbance values. Figure 2: Predictive graph depicting an exponential increase in the activity of integrase over time. Visualized using LacZ as a reporter gene. Negative, test, and positive controls correspond to the ONPG assay samples found in our results page.

Figure 2: Predictive graph depicting an exponential increase in the activity of integrase over time. Visualized using LacZ as a reporter gene. Negative, test, and positive controls correspond to the ONPG assay samples found in our results page.

Baseline Activity

Is this the natural activity of ΦBT1 serine integrase? Gathering more data will allow us to average the function of ΦBT1 serine integrase across multiple assays.

State Switchable Promoter Function

After ΦBT1 function has been characterized, the state switchable promoter (SSP) will be tested. BsmBI GGA will be used to assemble a multi gene plasmid from 2 modules: one containing a promotor, integrase, and the SSP, and the other containing GFP. Fluorescence will be normalized to ΦBT1 function and compared to a positive control made of a strong promotor directly upstream GFP. This will determine the efficacy of the forward SSP compared to a normal promotor.

Complete Memory Component

ΦBT1 integrase and the SSP will eventually be combined to form the memory circuit of seQUESTer.

Response Gene Testing Overview

Response Gene Testing

The end-goal of our regulatory genetic circuit is to turn yeast into hyperaccumulators of lead upon detection. Thus, genes selected for downstream response must be linked to the SSP, so their expression only occurs after the cell has detected lead. Genes will be expressed in an artificial operon-like system, dependent on the singular SSP. Viral 2A peptides placed at the 3' end of each transcript will stall translation machinery long enough for the nascent protein to fall off, terminating the sequence without the need of a stop codon. We have selected three genes to make up this response system; DMT1 as a transporter, GSH1 as a carrier, and YCF1 as a vacuole transporter. All three will be assayed prior to their insertion into the final genetic circuit.

Testing of Transporter Gene DMT1
Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) analysis

Wild-type BY4741 and BY4741 transformed with DMT1 will each be grown in lead supplemented media. The amount of lead up taken will be compared between cell lysates via Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES). Results will determine if DMT1 increases the rate of transportation of lead into the cell relative to diffusion across the plasma membrane in wild-type yeast. Yeast has a selectively permeable plasma membrane that may limit the absorption of heavy metals without an active transporter. Figure 3

Minimum Inhibitory Concentration (MIC) analysis

An MIC will be conducted to compare the tolerance of WT yeast and transformed yeast to Pb2+. Adding a transporter to increase lead-uptake could have adverse effects on yeast. An MIC will provide data to determine if adding DMT1 has adverse impacts.

Testing Sequestration Genes GSH1 and YCF1
Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) analysis

YCF1 is an ABC transporter that sequesters lead in vacuoles. It requires lead to be bound to a co-factor, glutathione (GSH1). These genes must be expressed in tandem for both of them to function. ICP-OES will be repeated on wild-type yeast, yeast containing a GSH1-YCF1 module, and yeast containing both DMT1, and GSH1-YCF1. The goal of this assay is to determine if cells transformed with GSH1-YCF1 can sequester more lead, and if the addition of DMT1 to GSH1-YCF1 makes this process more efficient.

Minimum Inhibitory Concentration (MIC) analysis

An MIC will be conducted to compare the tolerance of WT yeast, yeast containing GSH1-YCF1, and yeast with DMT1, and GSH1-YCF1. The results of this MIC will determine if the addition of GSH1-YCF1 helps increase yeast tolerance to lead by compartmentalizing this heavy metal in a vacuole. Figure 4

Testing of 2A Peptides

Viral 2A Peptides are used to sequentially pause translation in the response module, so that DMT1, GSH1, and YCF1 can all fall off as complete, separate proteins. Although this alternative to stop codons allows expression of response genes to be linked to the SSP, it may cause weaker translation of genes distal to the promotor.
This mechanism will first be tested by transforming yeast with a module containing pTEF2, followed by DMT1, GSH1, YCF1, and GFP. Genes will be separated by 2A viral peptides. The fluorescence of GFP will be compared to yeast transformed with a module containing GFP directly downstream of pTEF2. If a significant difference in fluorescence, or no fluorescence is observed, 2A peptides may need to be replaced with a multi-plasmid system, or shorter response genes may need to be used.
Additionally, these modules will be transformed and assayed in both haploid and diploid yeast strains. BY4741, a haploid yeast, was used for this project. BY4743 is a diploid variant from the same family as BY4741. If expression of response genes is insufficient in haploid yeast, transformation into BY4743 will double the production of these elements.

Full Circuit Testing Overview

Full Circuit Testing

The final phase of seQUESTer is to integrate our multi-gene plasmid into the yeast chromosome. The Deuber kit comes with a pre-designed vector with 5' and 3' sites homologous to the native URA3 locus in yeast. Figure 4
After integration, the half-life of yeast containing this genetic module must be characterized, to inform how long this organism remains viable. Additionally, we need to determine:

  1. Minimum and maximum levels of lead for detection
  2. The byproducts produced by transformed yeast, including potential hazardous metabolites
Once studied, this concept can apply to other pollutants. Its modular nature means any aptamer or response genes can be swapped in or out.