20.109(S24):M2D1

Introduction

Schematic of yeast H₂S precipitation model. Model system where yeast production of hydrogen sulfide precipitates metal from media, which can then be captured by yeast cell surface display. Adapted from Sun et. al. 2020. Nature Sustainability

Heavy metals are typically thought of as metal elements with a relatively high density. Many of these metals are considered precious (gold, silver, platinum) or have commercial value (lead, copper, nickel). Other metals can play an essential health role in small quantities (zinc, iron, manganese). However many of these metals are toxic, even when present in parts per billion. Heavy metals can be released into the environment through a number of activities such as mining, industrial production of consumer products, or disposal of electronic waste.

In this module, you will focus on protein engineering of a cell surface display peptide in Saccharomyces cerevisiae, also known as baker's yeast. We are using this YSD (yeast cell surface display) model to capture the heavy metal cadmium in a recyclable form. This type of protein engineering is an early step in optimizing a "bioremediation" model system. Bioremediation is an approach to cleaning up environmental pollutants by repurposing a known biological process. Typically, bioremediation utilizes microbial and fungal organisms to remove environmental contaminants as these single cell organisms can be fast growing and genetically tractable (i.e. relatively simple to genetically manipulate).

Today you examine what is known about our current S.cerevisiae bioremediation model system. You will explore the genetic metabolic engineering used to induce the yeast to produce hydrogen sulfide, examine the components of yeast cell surface display, and examine previously expressed peptides. You will use this information to rationally design a peptide to express on the yeast cell surface in order to capture Cadmium particles in the most uniform distribution possible. The rest of the module will be devoted to expressing your chosen peptides in our yeast model system, and testing these peptides for their suitability in bioremediation.

Protocols

Part 1: Review metabolic engineering approach to create ΔMet17 yeast

In this module,

Yeast metabolic pathway converting sulfate to amino acids. Met17 gene indicated in purple Adapted from Sun et al. 2020. Nature Sustainability

In your laboratory notebook, complete the following:

• How

Part 2: Review yeast cell surface display

Yeast cell surface peptide display with tags

In the

In your laboratory notebook, complete the following:

• Based

Part 3: Examine data for ΔMet17 yeast cell surface amino acids

Stability constants of cadmium with amino acids. Adapted from Sovago & Varnagy. 2013.

Now that you have noted relevant information regarding the ΔMet17 YSD model system, it's time to turn your attention to our metal of interest, cadmium. While there is limited information on the specific mechanism of cadmium capture by peptides, previous literature has indicated that amino acid residues form complexes of differing stability with cadmium. Using the table to the right, examine the reported affinities of cadmium for different amino acid residues. In previous work from the Belcher lab, the ability of particular amino acid residues to remove cadmium from spiked media was tested using yeast display. The results of that experiment are shown at right and detailed in Sun et. al, 2020 (linked on the Mod2 landing page).

Percent change in cadmium precipitation with amino acids. Adapted from Sun et al. 2020. Nature Sustainability

This data can be compared to the stability data in the table to identify amino acid residues that consistently bind cadmium. In addition to providing additional evidence of cadmium binding to amino acids, the data from Sun et. al was generated using a yeast display model very similar to the one you will employ.

In your laboratory notebook, complete the following:

• Which amino acids seem like likely candidates to capture precipitating cadmium?
• Which amino acids have lower affinity?

Cadmium precipitate particle patterns and fluorescent emission vary based on cell surface peptide sequence. Top row shows diffraction patterns of precipitated cadmium sulfate. Cuvettes show fluroescent emission of captured cadmium sulfide which is quantified on the right. More structured fringe lattices of cadmium sulfide precipitation result in stronger fluorescent emission. Adapted from Sun et. al. 2020

After looking at these data, you may be convinced that a string of cysteine residues are the most effective way to capture cadmium. This is indeed true. As the Belcher lab has found, a hexapeptide of cysteine repeats will effectively capture cadmium in solution. However, they also found that the cadmium sulfide particles which bind to cysteine peptide motifs form more amorphous cadmium sulfide deposits than a peptide motif which included glycine residues (Gly-Cys-Cys-Gly-Cys-Cys). We don't just want to capture cadmium with our model system. We want to capture it in a usable form, with crystalline features that produce a strong fluorescent signal.

Given this

Part 4: Choose a peptide sequence and determine a DNA sequence to encode it

Using the information you have gathered above, you can now determine what peptide you would like express in order to capture precipitating cadmium. ADD WAY MORE

Oligo sequences for amino acid codons. Image created in Biorender.

In your laboratory notebook, complete the following:

• What amino acid sequence have you decided?
• What DNA sequence have you chosen to encode your peptide?

Determine the DNA sequence you need to encode your amino acids and upload it to the Class Data page on the wiki before you leave.

• These primers must be ordered by 6pm to arrive in time for your next experiment.
• Anchor sequences will be added to your primers to orient the new sequence between the peptide tags. These sequences will be detailed in the next lab.

Navigation links

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