Development of Genetic Engineering Tools for The Iridescent Bacteria Cellulophaga Lytica

The study of <i>Flavobacteriacae</i>, a genus consisting of gram-negative, rod-shaped bacteria, has garnered increased interest, particularly in the field of synthetic biology, for their ability to break down complex organic matter as well as their gliding motility mechanisms. The primary focus of our group is to deepen our understanding of one such bacterium, <i>Cellulophaga lytica (C. lytica)</i>. Native to the rocky shores and mudflats of Costa Rica, this marine bacterium is capable of withstanding extreme variations in light, temperature, and salinity. One distinct feature of <i>C. lytica</i> is its unique, glitter-like, iridescence, which is believed to be due to the bacterium's ability to self-assemble into microstructures that reflect light at different wavelengths. The strain utilized in this project displays predominantly green iridescence, but red, orange, and purple can be observed as well. The genetic mechanisms governing displayed iridescence, however, remain understudied. It is suspected a specific cassette of genes (gld) is responsible for this observation. This study intends to display whether deletion of the GldB gene in <i>C. lytica</i> can disrupt the formation of uniform colonies, impacting biofilm formation and allowing us to control the iridescence patterns of the bacteria. This gene of interest was chosen for its known role in encoding proteins associated with gliding motility and cellular membrane morphology. If successful, this research will enable the construction of a genetic engineering platform using synthetic biology techniques that can disrupt, control or modify the gliding motility of <i>C. lytica</i>. Being able to harness this mechanism will allow for the incorporation of <i>C. lytica</i> into novel iridescent biomaterials and colorimetric biosensors for future military applications including soldier protection, promoting operational readiness and efficiently executing operations. This genetic toolbox could also be optimized for other marine bacteria as well.<br/><br/>Although biparental conjugation was also attempted, preliminary findings from our team support the use of electroporation as a transformation mechanism to deliver extraneous genetic material into <i>C. lytica</i> to interact with gldB. Testing and standardization of electroporation conditions were conducted with shuttle vectors pYT172 and pYT247, both of which contain the gene for erythromycin resistance (ermF). The growth of the <i>C. lytica</i> colonies on media plates supplemented with erythromycin was confirmed through sequential colony PCR and gel electrophoresis. Future modification of these shuttle vectors will be accomplished through Gibson/Golden Gate assembly, introducing an inducible promoter to regulate expression of a reporter protein (GFP). Alternatively, we also aim to design a suicide vector for target gene knockout of GldB. Essentially, by modulating genetic products that influence cellular communication, protein expression, and adhesion, controllable assembly and iridescence can be demonstrated in our model bacteria, <i>C.lytica</i>.<br/><br/>This foundation allows for further exploration into the feasibility of synthetic spatial patterning of the flavobacterium via 3D-bioprinting technology. Currently, our team is investigating the efficacy of exponential-phase liquid cultures encapsulated within varying concentrations of gelatin and sodium alginate as a form of “bioink”, leading to the formation of hydrogels upon crosslinking with 100 mM calcium chloride. Thus far, the idealized parameters for bioprinting and crosslinking while maintaining maximum cell viability have been achieved. Future endeavors aim to optimize the iridescence visibility within the hydrogel via genetic modification and altering precursor component ratios, unpacking the physiochemical characteristics of the hydrogel itself, and producing increasingly complex structures that are capable of being mobilized for use as colorimetric biosensors.