Contribution

Parts - Our S.thermophilus toolbox

We created several parts that function in our model chassis, S. thermophilus.

The first three allowed us to obtain fluorescent strains, enabling us to distinguish them under the microscope.

Then, we designed two parts enabling surface expression of proteins — respectively a nanobody and an α-rep. Both of these proteins recognize GFP, but at different epitopes. Therefore, when the nanobody is expressed in one fluorescent strain and the α-rep in another, an aggregation is promoted through GFP interaction and can be observed under an epifluorescence microscope.

We built a collection of these parts to make it possible to induce S. thermophilus aggregation via surface display. If you’re curious about the purpose of this aggregation and how it fits into our project, take a look below at the handbook we created on S. thermophilus — a fascinating chassis that we hope will spread within the iGEM community.

S. thermophilus Handbook

This handbook is designed to help future iGEM teams work with Streptococcus thermophilus. It contains background, protocols, and potential applications.

Preface

Our project is to create a bacterial production platform. To achieve this, we need a robust bacterial chassis: Streptococcus thermophilus. This Gram-positive bacterium is widely used in our host lab and is remarkably easy to manipulate. From the very beginning, many scientists were astonished by how convenient it is, especially for genomic editing.

These kinds of comments motivated us to write this handbook — a compilation of our refined protocols and methods, combined with expert advice. Our goal is to make S. thermophilus accessible to you, enabling research with just PCR and XIP.

Inside this handbook, you will find insights specific to our project: protein display on the cell surface, cell aggregation strategies, and, of course, precious guidance for working with Streptococcus thermophilus. We hope this handbook serves you well and becomes a valuable resource for your work.

Introducing S. thermophilus

Introducing S. thermophilus is challenging, but we focused on key generalities essential for understanding your future chassis: metabolism, genome architecture, and natural transformation.

Metabolism

Streptococcus thermophilus, our bacterial model, is a Gram-positive bacterium adapted for growth in milk and widely used in the dairy industry for yogurt production, which is worth approximately 40 billion USD worldwide [1]–[3]. First described by Orla-Jensen in 1919 [4], it is non-motile, non-spore-forming, catalase-negative, facultatively anaerobic, and homofermentative. Its natural habitats are restricted to the bovine mammary mucosa and raw milk. S. thermophilus is also the only member of the streptococcal genus to have GRAS (Generally Recognized As Safe) status [5].

The strain used here, LMD-9, is the most common in academic research. It shows regressive adaptation due to specialization in milk, particularly in sugar metabolism. LMD-9 preferentially uses lactose as a carbon source, cleaving it into glucose and galactose. The galactose is extruded in exchange for lactose import. It can also metabolize sucrose and fructose [6]–[9].

Genome Architecture

The most interesting aspect of S. thermophilus relates to genomic engineering. Before exploring this, it is necessary to understand some basics of the LMD-9 genome. It contains a 1,956,368 bp circular chromosome and two cryptic plasmids of 4,449 bp and 3,361 bp [5]. Out of 1,854 genes encoded in this nearly 2 Mb genome, 241 are pseudogenes, reflecting the specialized evolution of LMD-9 in milk. This includes frameshifted enzymes required for mannose and fructose utilization, which are otherwise common in other bacteria [10]–[13].

Natural Transformation

To modify this genome, we relied on the natural competence of LMD-9, regulated by the ComRS system [14], [15]. Briefly, ComS is expressed, processed, and secreted into the extracellular medium. Once externalized, ComS (called XIP, ComX-inducing peptide) is re-imported and binds ComR. The ComR–XIP complex dimerizes and acts as a transcription factor for ComX, which in turn activates competence genes [16].

Advantages and Disadvantages

Aggregation and Its Implications

For this project, there are three main reasons to study S. thermophilus aggregation: fermentation efficiency [17], quorum sensing efficiency [18], and division of labor [19], [20].

Since S. thermophilus grows under acidic and anaerobic conditions [21], it performs better at higher cell densities (Fig. 2). Individual cells actively deplete local oxygen via NADH oxidase activity [22]–[24] and increase acidity through lactic acid production [25], [26]. These conditions improve fermentation efficiency, highlighting the importance of controlling aggregation.

Regarding quorum sensing, it relies on the ComRS system, as described above for natural transformation (Fig. 3). Aggregation is relevant because XIP reinternalization likely requires cell-to-cell contact (Fig. 4), either due to rapid reintegration near the surface or localization at the membrane [18]. Controlled aggregation could trigger natural competence even at low cell concentrations or improve transformation yields.

For division of labor (DOL), the idea is to reduce the metabolic burden on a strain by dividing the pathway of interest between two or more strains [19], [27]. Without aggregation, synthetic communities face challenges: cheaters and diffusion [28], [29]. Diffusion of the metabolite of interest can be deleterious if (i) the product diffuses poorly, reducing exchange efficiency, or (ii) cheaters exploit the product, weakening the community [30], [31]. Cheaters often arise via mutation–selection processes [24]. Aggregation helps resist cheaters by ensuring preferential metabolite exchange among co-aggregates, which outcompete non-contributing strains [29], [30], [33].

S. thermophilus aggregation is thus highly promising: it is both an industrially relevant bacterium and a synthetic biology chassis with natural DNA uptake capability [8], [16], [32], [33]. Its simple carbohydrate metabolism also makes it a suitable chassis for sugar production that is not consumed by the cells.

Protocols

Protocol 1: Loxing of Streptococcus thermophilus

Materials and Reagents

• S. thermophilus strains carrying cassette to be loxed
• Chemically defined medium (CDM)
• Semi-skimmed milk (Campina brand or equivalent)
• M17G broth
• Selective antibiotics
• XIP peptide
• DMSO or glycerol (cryoprotectant)
• PCR reagents (for verification step)
• Incubators set at 30 °C and 37 °C
• Freezer at −80 °C

Procedure

Day −1

• Inoculate strains to be loxed in 1.3 mL CDM supplemented with the appropriate antibiotic.
• Incubate overnight at 37 °C.

Day 0

1. Transfer 50 µL of culture into 1 mL semi-skimmed milk.
2. Incubate for 1 h 15 at 37 °C.
3. Add 10.5 µL XIP carrying Cre plasmid and mix gently by inversion.
4. Incubate for 3 h at 37 °C.
5. Plate onto antibiotic-supplemented agar.
6. Incubate for 24 h at 30 °C.

Day 1

• Pick at least 8 isolated colonies.
• Resuspend each colony in 1 mL M17G broth without antibiotics.
• Incubate overnight at 37 °C.

Day 2

• Perform serial dilutions and plate on M17G agar without antibiotic (10⁻⁶ dilution).
• Incubate overnight at 37 °C.

Day 3

1. Select 10 colonies per isolate.
2. Streak each onto 3 plates:
   • without antibiotics,
   • with plasmid marker,
   • with lox cassette marker.
3. Incubate overnight at 37 °C.

Day 4

• Compare growth on plates.
• Inoculate M17G liquid medium with strains showing no resistance.
• Incubate overnight at 37 °C.

Day 5

• Add DMSO and store strains at −80 °C.
• Perform PCR to confirm loss of lox cassette.

Protocol 2: PEG/Ethanol DNA Purification

Materials and Reagents

• PEG 8000 (MW 6000–8000)
• NaCl
• Sterile ultrapure water (qH₂O)
• Ethanol 70% (cold, −20 °C)
• Ethanol 100% (cold, −20 °C)
• Centrifuge (12,000–14,000 rpm, RT or 4 °C)
• SpeedVac concentrator (optional)
• PEG solution (20% PEG 8000 + 2.5 M NaCl)

Preparation of PEG solution (50 mL):

• 10.0 g PEG 8000
• 7.3 g NaCl
• Add qH₂O to 45 mL
• Stir until PEG dissolves (may take >20 min). Use 37 °C incubator or water bath if necessary.
• Adjust to final volume of 50 mL with qH₂O.

Procedure

1. Add 0.6–1 volume of PEG solution to the PCR product (v/v).
2. Mix gently by inversion (5–10 times) or pipetting; incubate for 15 min at room temperature. Note: longer incubation (up to 2 h, without mixing) can increase yield.
3. Centrifuge 15 min at 12,000–14,000 rpm (RT or 4 °C).
4. Carefully discard supernatant without disturbing pellet.
5. Wash pellet with 5–10 volumes cold 70% ethanol.
6. Centrifuge 50 min at 13,000 rpm (RT or 4 °C).
7. Repeat steps 5–6 if higher DNA purity is required.
8. Remove ethanol completely; air-dry pellet 5–10 min at RT. Note: drying in a SpeedVac may improve DNA quality. Ensure no ethanol traces remain.
9. Resuspend DNA pellet in 20–50 µL ultrapure water.

Protocol 3: Epifluorescence Microscopy & Co-culture

Materials and Reagents

• Phosphate-buffered saline (PBS):
  • 137 mM NaCl
  • 2.7 mM KCl
  • 1.8 mM KH₂PO₄
  • 10 mM Na₂HPO₄·H₂O
• CDMg medium
• GFP (final concentration ≤5 µM)
• Agarose (1.5%) in PBS (for slide preparation)
• Centrifuge
• Incubator set at 37 °C

Procedure

Day −1

• Prepare PBS solution as described above.
• Start overnight cultures in 1.3 mL CDMg at 37 °C.

Day 0

1. Measure optical density of cultures.
2. Dilute into fresh CDMg to OD = 0.05.
3. Incubate 4 h at 37 °C.
4. Centrifuge cells, wash twice with 500 µL PBS, resuspend in CDMg.
5. Add GFP (final ≤5 µM) and incubate 2–3 h at 37 °C.
6. Centrifuge again, wash twice with 500 µL PBS, resuspend in 50 µL PBS.
7. Prepare microscope slides with 1.5% PBS–agarose and add 3 µL of cells.

Protocol 4: Preparation of Electrocompetent Cells and Electroporation

Materials and Reagents

• Bacterial strain of interest
• Growth medium (appropriate complex medium)
• Washing solution (ice-cold, composition adapted to organism)
• Electroporation solution (ice-cold, composition adapted to organism)
• Plasmid DNA (0.25 µg per transformation)
• Pre-chilled electroporation cuvettes
• Electroporator (set to 2,000 V, 5 ms time constant, prokaryote mode)
• Soft agar (pre-warmed)
• Selective agar plates with appropriate antibiotic
• Ice and ice bucket
• Sterile pipettes and tips

Procedure

Preparation of Electrocompetent Cells

1. Grow cells at 40 °C until an optical density OD₆₂₀ = 0.2–0.4 is reached.
2. Wash culture twice with ice-cold washing solution.
3. Resuspend cells in ice-cold electroporation solution to OD₆₂₀ = 1.7–1.8.
4. Keep cells on ice until electroporation.

Electroporation

1. Mix 0.25 µg plasmid DNA with 100 µL electrocompetent cells by gentle pipetting (do not vortex).
2. Transfer mixture into a pre-chilled electroporation cuvette.
3. Wipe cuvette dry and insert into the electroporator.
4. Apply pulse:
   • Mode: Prokaryotes
   • Voltage: 2,000 V
   • Time constant (τ): 5 ms
   • Expected result: up to 3.2 × 10⁴ transformants/µg DNA
5. Immediately add 1 mL ice-cold complex medium, resuspend cells, and transfer to a sterile tube.
6. Incubate 5 h at 40 °C for recovery.
7. Resuspend recovered cells in 4 mL soft agar and plate onto selective agar plates with antibiotic.
8. Incubate 2 days at 40 °C.

Protocol 5: Growth Media for Streptococcus thermophilus LMD-9

Materials and Reagents

• Streptococcus thermophilus strain LMD-9
• Skimmed Milk Medium (SMM)
• M17 medium
• Chemically Defined Medium (CDM) (composition according to Letort & Julliard, 2001)
• Lactose (1% w/v, for supplementation → M17L and CDML) or other sugars of interest (glucose, galactose, sucrose)
• Chloramphenicol (5 mg/L)
• Erythromycin (5 mg/L)
• Spectinomycin (250 mg/mL)
• 5-Fluoroorotic acid (5-FOA, 500 mg/L; stock prepared at 100 mg/mL in DMSO)
• Anaerobic incubation system (e.g., jars with anaerobic gas packs)
• Incubator set at 37 °C

Procedure

1. Prepare the following growth media as required:
   • SMM: Skimmed Milk Medium
   • M17L: M17 medium + 1% lactose
   • CDML: Chemically Defined Medium + 1% lactose
2. Add selective agents to the media at final concentrations:
   • Chloramphenicol: 5 mg/L
   • Erythromycin: 5 mg/L
   • Spectinomycin: 250 mg/mL
   • 5-FOA: 500 mg/L (from 100 mg/mL DMSO stock)
3. For solid media cultures: incubate S. thermophilus at 37 °C under anaerobic conditions.
4. For liquid cultures: inoculate cells into the appropriate medium and incubate statically (no shaking) at 37 °C.

Protocol 6: Transformation of Streptococcus thermophilus

Materials and Reagents

• S. thermophilus strain
• Chemically Defined Medium (CDM) with 1% glucose
• Semi-skimmed milk
• XIP (ComR-activating peptide), 500 µM stock solution
• DNA of interest (plasmid or linear fragment)
• M17 medium and M17-glucose (M17-Glu) agar plates
• Selection marker antibiotic (as required)
• Sterile 96-well plate
• Sterile glycerol (for cryostocks)
• Anaerobic incubation system (e.g., anaerobic jars with gas packs)
• Incubator at 37 °C
• Sterile microcentrifuge tubes

Procedure

Day −1

• Inoculate the strain in 1.3 mL CDM supplemented with 1% glucose.
• Incubate overnight at 37 °C.

Day 0

1. Transfer 50 µL of overnight culture into 1 mL semi-skimmed milk.
2. Incubate for 1 h 15 min at 37 °C.
3. Add 2 µL XIP (from 500 µM stock), mix gently by inversion (do not vortex).
4. Transfer 400 µL of culture into a sterile microcentrifuge tube.
5. Add 6 µL DNA of interest and mix gently.
6. Incubate for 3 h at 37 °C.
7. Prepare serial dilutions (10⁰ to 10⁷) in milk (15 µL into 135 µL milk, sterile 96-well plate).
8. Plate 100 µL of dilutions onto M17-Glu agar plates supplemented with selection marker.
9. Include a dilution control with a multichannel pipette (3 µL).
10. Incubate plates at 37 °C in an anaerobic jar.

Day 1

1. Pick 4 colonies and streak them on fresh plates (2 colonies per plate).
2. Incubate plates overnight at 37 °C in an anaerobic jar.

Day 2

1. Inoculate each confirmed clone into M17 broth.
2. Incubate at 37 °C.

Day 3

1. Prepare glycerol stocks: mix 800 µL culture with 800 µL sterile glycerol.
2. Store at −80 °C.

Our engineering of Streptococcus thermophilus

This part will help you to understand how we managed our project according to our chassis. How we took advantage of present proteins and specificities, making S. thermophilus the best chassis for this project.

a) Key hypothesis for protein display

Exopolysaccharides

- eps locus

The eps/cps cluster is composed of a group of genes directly involved in the biosynthesis of exopolysaccharides (EPS). This cluster has been identified in multiple strains and species of lactic acid bacteria, particularly in S. thermophilus. In general, the eps cluster of S. thermophilus ranges from 15 to 30 kb in size and is typically flanked by the deoD and bgIH genes. Many operons from eps clusters of different strains have been compared, leading to the proposal of a modular organization [34].

The first module (epsA–D) is involved in regulation and polymerization control, highly conserved across strains [35]. The second module, downstream, is variable and encodes glycosyltransferases and export machinery, essential for EPS biosynthesis. Interestingly, strain IP6756 lacks glycosyltransferase ORFs in its eps cluster, suggesting genes outside the locus may compensate [36].

- rgp locus

In addition to the eps cluster, the genome of S. thermophilus contains the rgp locus, predicted to be involved in extracellular polysaccharide production [34]. In S. mutans, homologous genes mediate the synthesis of rhamnose–glucose polysaccharides (RGP), determining serotype specificity (C, E, D) [37]. The conserved rgpA–F genes govern RGP assembly and export, while variable genes (rgpH, rgpI) control branching and diversity [38,39]. In S. thermophilus, the homologs of rgpA–F share high identity with S. mutans, but the variable genes are located upstream instead of downstream [34].

- hasAB locus

Hyaluronic acid (HA) is a high-molecular-weight polysaccharide composed of repeating disaccharide units of N-acetylglucosamine and glucuronic acid. The hasABC operon directs HA capsule biosynthesis, with hasA (hyaluronan synthase) and hasB (UDP-glucose dehydrogenase) sufficient for HA production [40–42].

Usage

In our project, exopolysaccharide usage relied on the deletion of the three loci (eps, rgp, hasAB). However, scarless deletions proved unstable: strains reverted to WT or duplicated loci. For example, rgp duplication and eps reversion were observed. Since these loci are highly variable, we recommend keeping the selection cassette or using inhibitors when working with ExoPS⁻ strains.

Htra

HtrA (STER_RS09790) is an extracellular housekeeping protease degrading misfolded proteins [43,44]. It contains an unstructured low-confidence region between its catalytic core and five transmembrane domains (predicted by AlphaFold). In the context of protein display, protease deletion is important to prevent degradation of constructs. However, ΔHtrA strains displayed reduced growth efficiency, possibly due to accumulation of misfolded proteins in the peptidoglycan. Combined deletion of HtrA and ExoPS⁻ proved co-lethal, highlighting their interdependent role in maintaining cell envelope integrity.

For these reasons, stay tuned for our results at the 2025 Grand Jamboree, where we will present our troubleshooting with ExoPS⁻ and ΔHtrA in full detail.

b) Building a Bacterial Display Based on Streptococcus thermophilus

To anchor a protein extracellularly, we used the endogenous PrtS enzyme construction. PrtS (annotated STER-RS04165) is a protease degrading casein [34], structured as follows: a signal peptide responsible for translocation and membrane anchoring, the core enzyme with its catalytic activity, and the CWSS (Cell Wall Sorting Signal) recognized by Sortase A. Sortase A cleaves PrtS from its membranal part at the LPXTG motif (between glycine and threonine) to covalently attach threonine to pentaglycine motifs on lipid II, precursors of peptidoglycan synthesis [45], [46].

The base of our construction is to replace the core enzyme of PrtS with our protein of interest. However, even with optimizations such as ExoPS- and ΔHtrA, constructs may still not be accessible to the extracellular medium due to the peptidoglycan barrier. To address this, we introduced a linker between the CWSS and the anchored protein. The choice of linker was inspired by AlphaFold predictions of HtrA, which revealed a disordered region between its catalytic moiety and membranal part, hypothesized to resist protease activity on the LMD9 cell surface.

Our constructions included PrtS amino- and carboxy-terminal parts, an HtrA-derived linker, and either α-rep BGFP or nanobody LaG16.

One improvement for S. thermophilus display would be to preserve more parts of PrtS. Later structural investigations revealed that PrtS could be divided into 4 main regions, including an endogenous linker hypothesized to resist peptidoglycan growth, leaving a smaller core enzyme accessible outside the cell.

• 1–37 = signal sequence
• 38–177 = disordered region
• 178–1288 = core enzyme
• 1289–1503 = endogenous linker
• 1504–1578 = disordered region
• 1578–1618 = CWSS (LPNTG motif)

The interaction of our proteins (nanobody LaG16 and α-rep BGFP) was modeled using PyMOL with PDB entries to assess binding sites and avoid structural clashes. These analyses confirmed their compatibility around GFP.

For future projects, we recommend preserving the endogenous PrtS linker, as it could significantly improve extracellular accessibility of displayed proteins.

c) Metabolic Engineering

As debated earlier, the simple carbohydrate metabolism offered by S. thermophilus can provide us with a substrate for metabolic interdependence. The first thing that came into our minds was to use galactose, half of lactose. As you remember, lactose is uptaken by an antiporter in exchange for galactose. We know that some strains of S. thermophilus do grow with galactose as a carbon source [47], but not LMD9.

By assuming that the galactose metabolization operon, pseudogenes annotated from STRE_RS06730 to STER_RS06750, inactive from regressive evolution in milk medium, can be “awaken” by random mutations, we plated LMD9 on M17gal plates and obtained the LMD9gal⁺ strain. The following step is to delete LacZ, annotated STER_RS06725, to obtain a strain unable to break down lactose but able to metabolize the galactose present in the medium, given by the WT strain. This could slow down lactose uptake, but lactose can also enter in exchange with a proton.

To delete LacZ, we first need to sequence the gal operon since mutations could have appeared within it and near the LacZ gene just upstream.

d) Division of Labor

Once your consortium is established, you are free to use any split metabolic pathway. As a proof of concept for our project, we planned to display proteins and chose to express a fucanase, degrading fucoidan (a fucose polymer) into fucose. This local fucose concentration near the partner strain increases uptake in S. thermophilus through unspecific sugar transporters.

The second strain of the consortium will then use this fucose to produce 2-FL (2-fucosyllactose), a fucosylated lactose [48].

You could implement any division of labor (DOL) you have in mind by leveraging our modular consortium design.

Collaboration with Bohemia Bio: GMO Regulatory Handbook Project Genesis

After the productive discussions held on the rooftop at the end of the Prague iGEM meetup, our team SyntCoLAB and Bohemia Bio found that we shared a common interest in understanding the intricate regulatory environment surrounding GMO-derived food products.

Aware of the difficulties many iGEM teams encounter when dealing with European biotechnology regulations, we decided to pool our expertise and create a valuable tool for the synthetic biology community.

The Importance of This Handbook

This collaborative handbook fills a crucial gap in iGEM resources. While synthetic biology presents great opportunities in the pharmaceutical and agricultural sectors, GMOs are complex biological systems carrying inherent risks that require careful regulation. The strict GMO policies of the EU present challenges for scientists and students who may not be prepared for the complexity of the regulatory landscape.

Our Approach

We identified two main obstacles that make it challenging to understand EU GMO regulations: regulations created in specific historical contexts that may be unclear to newcomers, and legal terminology that differs significantly from scientific language.

What You Can Expect

Our in-depth handbook addresses these obstacles through eight detailed chapters:

Rather than just listing regulations, we provide historical context and clear explanations to help make complex legal frameworks more understandable for the scientific community. This resource reflects our dedication to equipping future iGEM participants with the practical regulatory knowledge necessary for successful synthetic biology projects in Europe.

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