Difference between revisions of "Part:BBa K5477035"

Revision as of 03:30, 2 October 2024

UDPD-pGAL1/10-UGT2B15 detox module against BPA

This composite part consists of UDP-glucuronosyltransferase 2B15 (UGT2B15) BBa_K54770169 , pGAL1/10 bidirectional promoter BBa_K5477005 and UDP-glucose dehydrogenase BBa_K5477018. pGAL1/10 drives the expression of the two enzymes in the opposite direction. UDPD synthesizes the necessary precursor (UDP-glucoronic acid) that UGT2B15 uses for phase II detoxification of BPA (1) (2).

The composite part was cloned using the method of USER-cloning into YCp-H. YCp-H is a centromeric plasmid used in yeast that includes a HIS3 marker, allowing for selection in histidine auxotrophic yeast strains. Like other CEN plasmids, YCp-H contains a CEN sequence, ensuring that the plasmid replicates and segregates similarly to yeast chromosomes. This results in a low copy number (typically one to two copies per cell), providing stable maintenance of the plasmid.

Results

Objective: To evaluate the detoxification system by incubating it with specific contaminants and performing LC-MS analysis to determine whether new compounds are produced.

Methodology: The detoxification module - UGT2B15 - GAL1,10 – UDPD was induced with galactose and incubated with BPA. Following overnight incubation, each detoxification system was centrifuged in Eppendorf tubes to separate the cells from the supernatant. The supernatant, henceforth referred to as the “media” samples, was collected. Absolute ethanol was added to the cell pellets, which were vortexed with micro glass beads to lyse the cells. The mixtures were centrifuged to separate cellular debris from the lysate, and the resulting supernatant was collected as the “pellet” samples.

All samples were filtered prior to being loaded into LC-MS glass vials.

Result: We did not have optimized LC-MS methods available for detecting highly hydrophobic compounds, even with the use of a Reverse Phase Column. Additionally, it took some time to identify ethanol as a suitable solvent for the system available in our department. Therefore, the results presented here are based on data generated from a single run of the samples on the LC-MS system. A previous run, where DMSO was used as a solvent, was excluded from the experiment due to the presence of multiple nonspecific peaks.

Figure 1 Base Peak Chromatogram for UGT2B15 detox system. Pellet, media, control samples are included

The Base Peak Chromatogram for UGT2B15 detox system shows the peak for UGT2B15 yeast + BPA pellet (B8, light purple) between retention time 20 and 30 minutes. The same peaks are seen for empty yeast + BPA pellet (B10, light gray). These intesne peak look like hydrophobic parts of the cell debris.

Figure 2 Base Peak Chromatogram for UGT2B15 detox system zoomed in to 17.5 minutes to 21.0 minutes retention time.

Figure 3 Base Peak Chromatogram for UGT2B15 detox system zoomed in to 9.6 minutes to 10.8 minutes retention time.

The peak at retention time 10 minutes was one of the common peaks seen in all samples apart from BPA standard. This could be a signal given by yeast cells in general.

Sequence and Features

References

1. Hanioka N, Naito T, Narimatsu S. Human UDP-glucuronosyltransferase isoforms involved in bisphenol A glucuronidation. Chemosphere. 2008 Dec 1;74(1):33–6.

2. Oka T, Jigami Y. Reconstruction of de novo pathway for synthesis of UDP-glucuronic acid and UDP-xylose from intrinsic UDP-glucose in Saccharomyces cerevisiae. FEBS J. 2006 Jun;273(12):2645–57.