Part:BBa_K3765010:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

IISc-Bengaluru 2022

Measurement

We used superfolder GFP (BBa_K3765010) as a fluorescent reporter to measure the activity of our hypoxic promoters. Prior to activity quantification, we attempted to determine the basal change in fluorescence intensity that resulted from exposure to hypoxic conditions due to factors like global changes in protein expression and reduced folding efficiency that are independent of promoter activity. To do this, we used the strong constitutive EM7 promoter (BBa_K4278705) to drive sfGFP expression and quantified the change in fluorescence between aerobic and hypoxic cultures.

We first cultured TOP10 pP_EM7-sfGFP overnight under aerobic conditions in LB media at 37^oC and 180 RPM with the appropriate antibiotic. The stationary-phase culture was diluted to an O.D. of 0.80 in LB media fortified with 4mM cysteine to promote hypoxic growth. Hypoxic conditions were created in a glass tube and 2mL of this diluted culture was added to both the hypoxic glass tube as well as an identical aerobic glass tube that was capped, but not sealed. The hypoxic and aerobic cultures were incubated in the shaker-incubator at 37^oC and 120 RPM for six hours. 1mL of culture from each tube was taken into a microfuge tube and centrifuged at 3000 RPM for 2:00 minutes. 900uL of the supernatant was then removed and the pellet was resuspended in the remaining volume. 2uL of this was spotted onto a clean glass slide and covered with a cover slip. The slides were imaged with an Olympus fluorescence microscope at 100X magnification, with 5% emission intensity and 500ms exposure. Images were quantified using FIJI to measure the change in fluorescence intensity in individual bacterial cells between the aerobic culture and the hypoxic culture, averaged across ~30 bacterial cells.

Fig. 1 Difference in sfGFP fluorescence between E. coli grown under aerobic and hypoxic conditions

We imaged ~30 individual bacterial cells for each sample and quantified the sfGFP fluorescence intensity within the cell. The variation in intensity between cells in a sample was fairly tight, and the data suggests that sfGFP fluorescence is higher under normoxic conditions than under hypoxic conditions. Statistical analysis using Student’s t-test shows that this difference is significant with a p-value of 0.05, so the promoter-independent variations in sfGFP folding and global protein expression due to hypoxia must still be taken into account while using sfGFP as a reporter for hypoxic assays. Indeed, it has been reported that the sfGFP chromophore formation rate may decrease hypoxic conditions. The mean decrease in fluorescence observed was 33.93% compared to the normoxic baseline.

Prior iGEM teams have reported issues with fluorescent proteins folding incorrectly under low oxygen tension. Nevertheless, we can control for differences in fluorescence intensity that result from factors independent of promoter activity like the slower rate of sfGFP chromophore formation or by global changes in transcription under hypoxia. We can achieve this by normalising all measurements with respect to the measurements obtained when a strong constitutive promoter with no oxygen-sensitivity drives sfGFP expression under hypoxic and normoxic conditions. Activity can then be quantified by imaging single cells and measuring fluorescence intensity across a large number of cells in the presence and absence of oxygen.

The formation of the chromophore in GFP requires the presence of molecular oxygen to oxidise the α,β bond of residue 66, which is also the slowest step in the process. While GFP cannot be expressed in obligate anaerobes or in conditions of extreme hypoxia or anoxia, literature suggests that the formation of the chromophore is unaffected under mid-grade hypoxia (oxygen saturation of >0.06%). Thus, GFP (and by extension sfGFP) is an adequate fluorescent reporter for hypoxic conditions provided the experimental conditions do not approach complete anoxia.^[1]

References:

[1] Vordermark D, Shibata T, Brown JM. Green fluorescent protein is a suitable reporter of tumor hypoxia despite an oxygen requirement for chromophore formation. Neoplasia. 2001 Nov-Dec;3(6):527-34. https://doi.org/10.1038/sj.neo.7900192. PMID: 11774035; PMCID: PMC1506559.