Background

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Chromoproteins, commonly used in biological research, serve as essential markers, with green fluorescent protein (GFP) being one of the most well-known example due to its fluorescence and versatility in imaging (Tian et al. 2023). Beyond their role as biological markers, chromoproteins, in their broader definition as any colored proteins, have garnered increasing interest for their potential applications in various industries, such as bio-dyes. The demand for sustainable alternatives in textile dyeing has driven research into natural, bio-derived dyes, including chromoproteins.(Pranta & Rahaman 2024) Leveraging Uppsala University’s expertise in chromoprotein research, including contributions from previous iGEM teams and a published study by a Uppsala-based research group, we embarked on the challenge of identifying new chromoproteins suitable for use as bio-dyes.

While our primary focus has been on chromoproteins for textile dyeing, we acknowledge their broader potential applications, which we explored in our Human Practice initiatives.

We began our work with some 40 environmental strains sourced from Brandis Lab at ICM, Uppsala University, a majority of which had not been fully sequenced. Initial efforts focused on investigating whether known chromoproteins could be detected within these strains. Following comprehensive strain characterization, we selected ten strains that demonstrated robust growth and color retention in both solid and liquid media for further study. The selection was based on the practical need to focus on strains that thrive in liquid medium, facilitating practical biochemical analysis of the colored proteins.

The selected strains were subjected to cell lysis, during which we optimized lysis protocols to accommodate the distinct characteristics of the strains. Post-lysis, three strains exhibited color in the supernatant, suggesting the presence of chromoproteins. Size exclusion chromatography (SEC) was employed alongside pigment assays to differentiate chromoproteins from other pigments. This approach, inspired by previous work in the field, yielded two candidate strains with potential chromoproteins.

Subsequent concentration using spin columns and semi-native SDS-PAGE analysis of the candidate strains, alongside known chromoproteins, revealed a yellow band that may indicate the discovery of a novel chromoprotein. Additionally, nonpolar separation techniques were applied to strains that retained color in the pellet but not in the supernatant, exploring the possibility of membrane-bound chromoproteins.

To support our experimental findings, a bioinformatic analysis was conducted to identify chromophore-related genes in strains with full sequencing data. Interestingly, the two strains selected for SDS-PAGE analysis corresponded to bioinformatic predictions by their Chromosearch pipeline, strengthening the hypothesis of chromoprotein presence. The predicted protein sizes, while approximate due to semi-native conditions, provided a reasonable basis for further investigation.

Experimental methods

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From plate to liquid

To assess the potential of the environmental strains provided by Brandis Lab at ICM, we began by submitting a Check-In form to iGEM, as most of the strains were not on the approved white list. After receiving approval for some of the strains, we selected 17 to continue working with.

We initiated the process by restreaking each strain onto fresh LA agar plates to observe their growth rates and determine when they started to produce color. Simultaneously, liquid cultures were prepared by inoculating the strains into liquid LB medium. As reference we conducted the same steps on E.coli strains containing known chromoproteins amilCP and mRFP1.

Lysis from liquid cultures

We continued working with the 10 strains that exhibited both growth and color in liquid LB medium. Liquid cultures were prioritized to allow for effective lysis of cells in this liquid medium as compared to non-liquid cultures. Initially, a standard lysis protocol using lysozyme, typically applied to E. coli, was implemented. This protocol was tested on the environmental strains as well as reference E. coli strains containing known chromoproteins, specifically amilCP and mRFP genes. Only a few of the environmental strains were successfully lysed in the first attempt. To address this, we optimized the protocol by extending the time for centrifugation and increasing the centrifugation speed, and incorporating a freeze-thaw step.

Size exclusion chromatography experiments

To further investigate the chromoprotein production in our strains, we focused on the three strains that exhibited good yellow color in the supernatants: 91, 153, and 350. Our goal was to remove any unwanted particles from the lysate supernatants and gain insight into the source of the observed color. Drawing on our prior education and a lecture on separation methods by Professor Gunnar Johansson, we identified size-exclusion chromatography as the most suitable method. At our disposal were Cytiva’s PD-10 desalting columns, pre-packed with Sephadex G-25 resin, which offered a cut-off of 5 kDa. This cut-off was deemed appropriate for our intended application of isolating the chromoproteins.

We ran some tests first with mRFP1, amilCP and some known small pigments. Here we could see how the small molecules, such as bromophenol blue, bound to the column while the proteins mRFP1 and amilCP were eluted. We then performed the same tests with our lysate supernatants of strain 91, 153 and 350. For 91 and 350 colored bands could be eluted, leading us to hypothesize that the color was, or was bound to, a particle large enough to be a protein. We discontinued the work with environmental sample 153 since the colored substance from this strain could not be eluted from the lysate.

Concentration

For environmental strains 91 and 350 350 our work continued to characterize the eluate from the PD10 columns. The eluate was now cleaned of particles and molecules not linked to the colored substance. Due to dilution of the band in PD-10 chromatography, we proceeded to concentrate the samples in preparation for SDS gel analysis.

After doing some research and carrying out discussions within the team there were a few directions we could go in (Goldring 2019), but most practical for us was to use protein concentrator spin columns. We did not know the size of our desired protein, so we went for a column with low molecular weight cut off at 3kDa to avoid accidentally losing the color. After centrifuging the samples in the spin columns, clear liquid was removed and we had a smaller volume of strongly colored samples to move on with.

Semi native SDS-gel analysis

Following concentration, eluates from the two environmental strains 350 and 91, were prepared for semi-native SDS-PAGE analysis. Precast 12% polyacrylamide SDS-PAGE gels were used together with a standard SDS-running buffer. The samples were not treated according to conventional SDS sample preparation protocols, instead, they were maintained in their native state to preserve both structure and color. Loading dye lacking beta-mercaptoethanol and SDS was employed to prevent denaturation of potential chromoproteins.

The samples were loaded alongside the known chromoprotein mRFP1, which served as a reference, as well as protein ladders. It is important to note that size estimations are approximate due to the non-denatured state of the proteins. Electrophoresis was conducted at 100V and 0.5A until the loading dye had fully migrated through the gel, taking approximately 90 minutes.

It is also noteworthy that when similar gel assays were performed with chromoproteins of the GFP family, they retained their secondary structures and oligomeric states, as evidenced by the color in the gel (see Fig.2 Bao et al. 2020).

Long term storage

At the end of our lab time, the promising bacterial strains (91 and 350) in liquid culture were mixed with glycerol, then frozen with a final concentration of 20% glycerol, and stored in the -80°C freezer for potential future experiments.

Pellet nonpolar separation

Reading some articles about pigment extraction from bacteria, using solvents to extract seems to be the most common first step (Rajendran et al. 2023) (Venil et al. 2014) . This is assuming that the color is from a non-water-soluble pigment. We had many colorful pellets from the lysing process and supernatants that were clear, indicating that the color may not be water soluble. Our background research on our strains also made us believe we could be working with some nonpolar pigments.

Based on the information we had, we wanted to resuspend the pellets from the lysates of strains 91, 108, 146, 149, 156 and 173 in both acetone and ethanol. Many of the samples did seem to contain pigments that could be suspended in ethanol, acetone or both (see table below). Then we tried to evaporate the ethanol and acetone, but due to time limits, only the acetone successfully evaporated.

References

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Bao L, Menon PNK, Liljeruhm J, Forster AC. 2020. Overcoming chromoprotein limitations by engineering a red fluorescent protein. Analytical Biochemistry 611: 113936.

Goldring JPD. 2019. Concentrating Proteins by Salt, Polyethylene Glycol, Solvent, SDS Precipitation, Three-Phase Partitioning, Dialysis, Centrifugation, Ultrafiltration, Lyophilization, Affinity Chromatography, Immunoprecipitation or Increased Temperature for Protein Isolation, Drug Interaction, and Proteomic and Peptidomic Evaluation. I: Kurien BT, Scofield RH (red.). Electrophoretic Separation of Proteins: Methods and Protocols, s. 41–59. Springer, New York, NY.

Pranta AD, Rahaman MdT. 2024. Extraction of eco-friendly natural dyes and biomordants for textile coloration: A critical review. Nano-Structures & Nano-Objects 39: 101243.

Rajendran P, Somasundaram P, Dufossé L. 2023. Microbial pigments: Eco-friendly extraction techniques and some industrial applications. Journal of Molecular Structure 1290: 135958.

Tian F, Xu G, Zhou S, Chen S, He D. 2023. Principles and applications of green fluorescent protein-based biosensors: a mini-review. Analyst 148: 2882–2891.

Venil CK, Zakaria ZA, Usha R, Ahmad WA. 2014. Isolation and characterization of flexirubin type pigment from Chryseobacterium sp. UTM-3T. Biocatalysis and Agricultural Biotechnology 3: 103–107.