Part:BBa_M50497:Experience

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Applications of BBa_M50497

This device is to be used in Escherichia coli to detect either endogenous or synthesized uvrY and express GFP upon binding of uvrY to the promoter sequence. Parts Bba_M50491 and Bba_M50494 are incorporated into this device.

User Reviews

UNIQ6aa18957f62f8831-partinfo-00000000-QINU UNIQ6aa18957f62f8831-partinfo-00000001-QINU

Our designed DNA fragments were ordered from an external company and synthesized with the BioXp Gibson Assembly Technology.

An initial assay was conducted to test the presence of fluorescence. Cultures of E. coli harboring plasmids luxSp2, luxSp1+sp2, and pColi (a plasmid including a rhamnose inducible promoter driving GFP production and an ampicillin resistant gene) were grown in EZ Rich Media with 12 mL 20% glucose and ampicillin for 16 hours and were each then diluted to an OD600 of 0.01.Of these samples, 1 mL triplicates of each culture were compiled in a 96-well plate and grown in EZ Rich Media in a 37℃ shaking incubator for two days. A pColi-harboring culture without rhamnose served as the negative control, as no fluorescence was expected, while a pColi-harboring culture with 1 mM rhamnose served as the positive control, as GFP expression is maximally rhamnose-inducible under this concentration. After incubation, 200 uL of each sample were transferred into a clear 96-well plate for OD600 and GFP (400 nm/515 nm) measurements using a plate reader.

Our results demonstrate that both Bba_M50497 and Bba_M50505 produce some amount of GFP and, as such, all contain endogenous uvrY as well. As seen in the figure below, the luxSp1+Sp2 plasmid produced the most amount of fluorescence, and consequently GFP, when examined through the microplate fluorescence reader. With this data, we concluded that the dual-promoter sequence contained within the Bba_50497 is more sensitive to endogenous uvrY compared to the single promoter construct, Bba_M50497.

To corroborate GFP production, an SDS-PAGE and Western Blot assay were conducted on the two plasmids along with the same positive and negative controls as above. The positive and negative controls were the same as in the fluorescence assay above. Two Western Blots were conducted in which either one or both Comet GFP antibody (7.5 uL Thermo-Fisher Alexa Fluor 488-conjugated anti-GFP antibody19 in 10 mL Blotto in PBS with Azide) and 6XHIS Tag primary (at a concentration of 1:2000) and secondary antibodies (anti-mouse HRP secondary antibody incubation at a concentration of 1:5000) were used in the appropriate procedures.

Utilizing a 6XHIS Tag primary antibody and an anti-mouse HRP secondary antibody, our membrane showed bands on the luxSp1+Sp2 plasmid. The bands for luxSp1+Sp2 corresponded to those anticipated for GFP, which has a length of 27 kDa. Thus, Bba_50497 underwent successful uptake by transformed E. coli and effectively produced GFP, indicating endogenous uvrY presence. Bands were not observed on the positive and negative control, which was expected because the previous assay revealed minimal GFP production. Additionally, no bands appeared for Bba_M50505 wells, revealing either that (1) transformants did not successfully uptake the luxSp2 plasmid or (2) transformed E. coli is not producing sufficient GFP for detection.

We also set out to test the effects of hydrogen peroxide on endogenous uvrY production. The same cultures used for the initial GFP assay were grown in EZ Rich Media with 12 mL 20% glucose and ampicillin for 16 hours and then diluted to an OD600 of 0.01. Of these samples, 1 mL triplicates of the two experimental and two control groups previously described were compiled in a 96-well plate and grown for a two-day period in a 37℃ shaking incubator. Increasing concentrations of hydrogen peroxide (0 mM, 0.25 mM, 0.5 mM, 1 mM, and 2 mM) were added to samples. After incubation, 200 uL of each sample was transferred into a clear 96 well plate for OD600 and GFP (400 nm/515 nm) measurements using a plate reader.

We observed that increasing hydrogen peroxide concentration to 2 mM drastically increased levels of GFP expression among all samples. From these results alone, it can be assumed that higher levels of GFP might be indicative of higher levels of endogenous uvrY, promoted by increased hydrogen peroxide concentrations. Furthermore, comparing Bba_M50497 and Bba_M50505 in each of the hydrogen peroxide concentrations reveals that the latter tends to slightly outperform former in hydrogen peroxide levels of 0.25mM and 0.5mM, but this trend changes when concentrations become 1mM and 2mM where the former outperforms the GFP production of the latter.