| NDSU - iGEM 2022

Initial Results

After working through many different constructs, we were able to successfully produce several cultures that were both red and had the cellulose binding domain present within the open reading frame. There were noticeable differences in vibrance across different parts after pelleting overnight cell cultures.

Constructs 50 and 60 both contained only mScarlet with a 6x-His tag in the open reading frame. The visual differences in vibrance show that the arabinose operon ((6)) resulted in significantly higher expression than the high strength constitutive promoter (J23102).

Constructs 46 and 49 both contained a cellulose binding domain in their open reading frame. 46 has the CBDfimi domain and 49 has the CBDclos domain. The differences in vibrance between these two strains shows that the choice of cellulose binding domain either affects overall expression or the vibrance of an individual protein.

Construct 48 contained the chromoprotein meffRed and CBDfimi , which similar to previous attempts, resulted in no color.

These initial results are confirmed with verified plasmids sequences from PlasmidSaurus seen below.

Cell pellets of various E. coli cultures containing our recombinant protein constructs. Each part corresponds to the open reading frame shown above.

Plasmid constructs pGEC046, pGEC048, pGEC049, pGEC050, and pGEC060 visualized in Geneious software for ease of comparison.

Cell Lysis

Samples 46 (left), 49 (middle), and 50 (right) after lysing cells and centrifuging insoluble material.

After lying our cell cultures with B-PER, lysozyme, and DNASE I, and centrifuging the remaining insoluble material, it was clear that a vast majority of our protein was sequestered into insoluble inclusion bodies. This makes sense as in order to visually see color in the colonies, even cell cultures with inducible promoters needed to be grown overnight.

Visual Results

Due to time constraints, we were unable to purify our cell lysate, determine concentration, or determine the dissociation constant of our fusion protein. However, we wanted to qualitatively show that our constructs were better at binding to cotton than the mScarlet negative control as a preliminary result. To make a direct visual comparison, the mScarlet negative control was diluted 1:16 to give the same fluorescence as the fusion protein. Applying the samples to a t-shirt produced results too transparent to see, showing that a much higher concentration is needed for the dye to be commercially viable.

Cotton t-shirt after directly applying 1mL of each sample.

Despite this shortcoming, we still wanted to determine if the cellulose binding domain was functional. Since our proteins exhibit fluorescence, we could image dyed t-shirt swatches with a trans-illuminator. Three replicates of both sample 46 and 50 were created by dyeing cotton t-shirt swatches with 1mL of the respective solution and placing them in falcon tubes at room temperature overnight. The following day, the samples were imaged on a trans-illuminator before washing. Washing took place by adding 40mL water to each tube and shaking at 250rpm for 30 minutes. Images were then taken after washing to show if the cellulose binding domain helped mScarlet stay attached to the cotton.

Experimental results of the wash experiment. Pre-wash (top left), post-wash (top right), pre-wash with reduced brightness (bottom left), and post-wash with reduced brightness (bottom right).

Quantitative testing after purification of our protein is required to definitively show that the cellulose binding domain is functional. However, the results from this experiment suggest that it is functional, even in the presence of the B-PER detergent used for cell lysis.

Conclusions

A successful fusion protein between mScarlet and a cellulose binding domain was created and verified with sequencing.
The choice of cellulose binding domain and chromoprotein is important in overall function because pGEC049 was visibly less vibrant than pGEC046.
Due to the necessity of producing large amounts of protein to be visible as a dye, inclusion bodies will likely be present. As a result, inclusion body purification must be taken into account in large scale production.
All three of the pGEC046 replicates showed a successful initial staining before the wash, but the pGEC050 replicates struggled even with initial staining. This implies that the cellulose binding domain present in pGEC046 is important in the initial binding to cotton.
The pGEC046 replicates retained more color after the wash. The third replicate of pGEC050 showed a clear loss of color after its wash, while the 3 replicates of pGEC046 showed retention of color. This indicates that pGEC046 is more resistant to washing and that the cellulose binding domain is important for permanent staining.

Future directions

There are many more areas to work on in order to make Dyenamix a reality. A few of these objectives include:

Solubilizing inclusion bodies and then purifying each construct through IMAC chromatography for quantitative testing
Determining the dissociation constant between each construct and its cellulose substrate
Quantifying a necessary protein concentration for commercial dyeing
Performing directed evolution assays to increase vibrance of successful constructs
Determining persistence of dyed fabric under various washing procedures
Creating new constructs with different colors