COFACTOR BASED FLUORESCENT PROTEIN FOR NEW OXYGEN-INDEPENDENT METAL SENSORS

The gastrointestinal microbiota is closely related to human health. Many of their metabolisms require the uptake of trace metals from the host. Current metal sensors are derived from green fluorescent proteins (GFP), which require oxygen to develop their fluorescence, therefore, they cannot be applied to study the anaerobic environment. The Zastrow lab is developing a new oxygen-independent zinc sensor by combining the CreiLOV domain from Chlamydomonas reinhardtii and the GAF3 domain from cyanobacteriochrome (CBCR) using a zinc binding domain. Site-directed mutanogenesis was conducted on CreiLOV to help abrogate the photocycle which results in the loss of its fluorescence. Next, plasmid cloning by PCR was used to transfer the gene from mammalian to bacterial vectors for expressing in E. coli. DNA sequencing results confirmed the plasmid has been made. The level of expression was then estimated by the solubility screening diagnostic to determine the best condition for the gene to be purified. The condition at 16°C, 0.5 IPTG, OD600 ~ 0.5 seems promising, however, more tests will be considered to achieve the highest result. The protein will be ready to be purified and characterized by UV-Vis and fluorescent spectroscopy in the future works.