1. Synthesize a 2433 bp DNA sequence, with PciI and EcoRI restriction sites at the 5' and 3' end respectively. For exact sequence, see appendix. The sequence map can also be seen on the design page.
2. Digest the pREST/EmGFP_verA vector (obtained from AddGene) with PciI and EcoRI restriction enzymes. Also, digest the synthesized DNA sequence from Step 1 with PciI and EcoRI.
3. Run digested DNA samples on a gel. Extract and purify the larger fragment from the digested vector and the digested synthesized DNA from the gel.
4. Ligate the gel recovered fragments using T4 DNA ligase to reconstitute the plasmid.
5. Transform the plasmid into DH5α competent cells using heat shock.
6. Plate transformed cells onto LB Ampicillin agarose plate and allow to grow overnight inside the incubator.
7. The next day, pick 10 single colonies from the agarose plate, grow them individually in 5 mL LB Ampicillin liquid culture overnight.
8. Extract plasmid DNA from each culture using a plasmid miniprep kit.
9. Digest purified plasmid DNA with PciI and EcoRI. Run digested DNA on a gel and look for the presence of 2 bands in the sample: 3378bp and 2433 bp. This would provide a preliminary confirmation of the successful cloning of our genetic construct. Ideally all work should be confirmed with sequencing (but as a high school team this might not be feasible).
10. Culture the positive clones on a larger scale and refrigerate appropriately for later usage in experiments.
1. Prepare 1 mL of pure water, 1 mL of 100,000 ng/L oxybenzone, and 1 mL of 100,000 ng/L estrogen. Add them separately into 3 tubes of 5 mL of transformed E. coli cultures made in step 10. Culture for 2 hours. The water acts as a negative control while the oxybenzone and estrogen and positive controls.
2. Measure absorbance of GFP with spectrophotometer, at excitation 488 nm/emission 510 nm.
The expected result is that the fluorescence intensity of oxybenzone and estrogen samples will be similar (as they are our positive controls).
It is also expected that there will be a significant difference in fluorescence intensity of GFP signal between pure water (serving as negative control) and the 100,000 ng/L oxybenzone and 100,000 ng/L estrogen.
If there is no difference in expression between the pure water and oxybenzone/estrogen samples, then we will have to go back to the design of the plasmid or carry out troubleshooting steps.
Many of our references used mammalian systems so the adaptation to bacteria may have caused issues in expression of our sensory protein (estrogen receptor). We could try the same system in a eukaryotic cell (like yeast) to see if there is a change or if the issue is fixed - if not the error lies further upstream.
We could also try to optimize the expression levels of our genetic constructs by the following methods:
1) Better optimize the transcription elements of our prokaryotic system by integrating sequences like the pribnow box (analogous to the eukaryotic TATA box).
2) Codon optimized the sequence for our proteins of interest for better expression in bacteria.
3) Look into protein modeling to identify if the estrogen receptor expression and functioning requires any specific post-translational modifications which may be absent in the E. coli.
1. Collect sea water from a remote area.
2. Prepare a 100,000 ng/L sample of oxybenzone and of estrogen using collected sea water.
3. Repeat system validation #1 with sea water samples.
The expected result is that the fluorescence intensity of oxybenzone and estrogen samples are similar. It is also expected that there is a significant difference in fluorescence intensity of GFP signal between just sea water and the 100,000 ng/L oxybenzone or 100,000 ng/L estrogen sample.
An unexpected and possible result would be the sea water sample having a significantly higher fluorescence intensity than pure water or no significant difference between the sea water and the oxybenzone/estrogen. This would indicate that there are already high levels of estrogen or oxybenzone present in the sea water sample being tested.
This is very unlikely to happen as, based on our research, areas with less traffic should have oxybenzone levels significantly less than 100,000 ng/L [1]. In addition, our research also showed that sea water from remote areas has almost undetectable levels of estrogen (<0.04 ng/L) [2] and should have readings similar to our negative control of pure water.
1. Create a 5 step serial dilution with oxybenzone concentrations of 100,000 ng/L, 10,000 ng/L, 1,000 ng/L, 100 ng/L, and 10 ng/L dissolved in collected remote sea water.
2. Add 1 mL of each dilution separately into tubes of 5 mL of our transformed E. coli cultures made in step 10. Culture for 2 hours.
3. Measure fluorescence intensity of GFP with spectrophotometer, at excitation 488 nm/emission 510 nm.
4. Use fluorescence intensity to create a standard curve corresponding to oxybenzone concentration and GFP fluorescence. The LoD is found at the highest concentration that absorbance is equal to that of remote sea water.
1. Collect local sea water from 5 different areas.
2. Add 1 mL to 5 mL of transformed E. coli cultures made in step 10. Culture for 2 hours.
3. Measure fluorescence intensity of GFP with spectrophotometer, at excitation 488 nm/emission 510 nm.
4. Use the standard curve made in the previous experiment to calculate the oxybenzone concentration associated with that particular fluorescence reading.
[1] Summary of oxybenzone near-reef water column concentrations globally ... (n.d.). Retrieved October 8, 2022, from https://www.researchgate.net/figure/Summary-of-oxybenzone-near-reef-water-column-concentrations-globally-Left-Box-plots_fig1_348972671
[2] Estrogens from sewage in coastal marine environments. (n.d.). Retrieved October 9, 2022, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1241440/