Difference between revisions of "Part:BBa M50006:Experience"

Revision as of 01:41, 12 December 2016

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.

Applications of BBa_M50006

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 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 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.

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. We used a deep well plate to leave our lead concentrations and bacteria cultures in the shaking incubator overnight for longer exposure. And consequently, we diluted our bacteria 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. Also, we expected to see either constant OD600 readings across our trials or a decline in OD600 readings at the 5mM and 10mM concentrations due to toxicity. Constant readings would indicate that the cells were grown up in the first place and died. On the other hand, low OD600 values would indicate that the lead concentration was so immediately toxic that no cell growth occurred.

User Reviews

UNIQ269a871933cb0f6c-partinfo-00000000-QINU UNIQ269a871933cb0f6c-partinfo-00000001-QINU

@@ Line 6: / Line 6: @@
 ===Applications of BBa_M50006===
 [[File:ZrapTimeNew.jpg]]
+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 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 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.
 [[File:ZraPToxicity.jpg]]
+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. We used a deep well plate to leave our lead concentrations and bacteria cultures in the shaking incubator overnight for longer exposure. And consequently, we diluted our bacteria 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. Also, we expected to see either constant OD600 readings across our trials or a decline in OD600 readings at the 5mM and 10mM concentrations due to toxicity. Constant readings would indicate that the cells were grown up in the first place and died. On the other hand, low OD600 values would indicate that the lead concentration was so immediately toxic that no cell growth occurred.
 ===User Reviews===