Part:BBa_M50006: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.

Stanford Location

Glycerol Freezer Stock Information:

Plasmid name: zraP; DNA 2.0 Gene ID: 273920; Organism: E. coli; Device type: promoter; Barcode #'s: 0133027169 (Marilu), 0133023922 (Janelle), 0133023904 (Katie); Box label: BIOE44 F16

Applications of BBa_M50006

Figure 1. Normalized GFP of E. coli + zraP over 6 hours

This assay measured the response time of our engineered E.coli to varying lead ion concentrations. We measured GFP fluorescence and OD600 every hour for six hours. We created lead ion concentrations of 0mM (negative control), 0.5 mM, 1mM, 1.5 mM, and 2 mM from the stock 100mM solution we made with dH2O. However, after seeing our inconclusive results and discussing with TAs and professors, we realized that diluting our lead concentrations with dH2O, rather than LB which we used as a blank, may have watered down our experimental readings. Our genetically modified E. coli + zraP did not exhibit the fluorescence results we expected. We hypothesized that it should respond to increasing lead concentrations over time depending on how sensitive zraP is to lead. Instead, we saw that GFP outputs oscillated as time increased, with a general decrease in the smaller concentrations and then a general increase in the larger concentrations. We did not see any changes over time, most likely because the E. coli + zraP was not given enough time to activate. Overall, the normalized GFP values did not become positive in the first 6 hours, which is paradoxical as we would expect our values to either be equal to or higher than our GFP measurement for our blank. These conclusions led us to consider that our lead concentrations were not high enough for activation, and we decided to try studying our construct under higher lead concentrations in our next assay.

Figure 2. Our results for Toxicity Assay #2. pColi and E. coli normalized GFP values initially decrease with small concentration, whereas zraP initially increases.

In our Toxicity Assay, we intended to measured how low or high of a lead ion concentration is needed to make our engineered E. coli glow fluorescently without dying. We created a new 100mM stock solution with LB, and made new lead ion concentrations, adding 5mM and 10mM to test for toxicity as well as 0.25mM and 0.75mM with the intent of capturing the dose-response curve trend around this more sensitive concentration range for fluorescence response. We used a deep well plate to leave our lead concentrations and bacterial cultures in the shaking incubator overnight for longer exposure. And consequently, we diluted our bacterial cultures (1 mL each culture, 9 mL LB) in order to create space for the cells to grow overnight. Overall, we expected to see increasing fluorescence as concentration increased, with a drop when concentration levels became toxic. According to the graph, pColi and E. coli normalized GFP values initially decrease with small concentration, whereas zraP initially increases. However, although our results do not show the S-shaped dose-response curve, we can still conclude that these results are potentially promising for zraP detecting harmful levels of lead in contaminated water at these critical lower concentrations near the EPA standard (15 parts per billion, or 7.24x10-11 mM) [6]. However, we also see that zraP had the lowest overall normalized GFP expression, although we expected it to have more fluorescence expression than the plain E. coli. We see that the normalized GFP values for zraP become negative starting around 2mM, which signifies that the cells started to die at this concentration and not express fluorescence. This points to a potential critical concentration threshold for toxicity in zraP.

User Reviews

UNIQ04dea4d4cfa563b1-partinfo-00000000-QINU UNIQ04dea4d4cfa563b1-partinfo-00000001-QINU