Team:USNA Annapolis/Demonstrate

Demonstrate

Showing a Working Project

Full Construct Testing

Graphs of Testing

Although we were happy to see colonies on our full construct plate, we were not so happy when we tested these colonies for the expression of GFP. Although we did see some resemblance of a relationship between the amount of sodium and the expression of GFP, the data seemed to be a bit problematic. We took a few of our full construct colonies as well as a few wild type Kanamycin resistant colonies and we inoculated into overnight culture tubes and used to determine if the Na+ sensor triggered the expression of GFP when exposed to increasing concentrations of sodium chloride. The assay was performed in a multi-well plate format inside a plate reader at an absorbance of 600 nm. The hypothesis was that the more sodium we add, the higher the absorbance would be for the expression of GFP. This was not quite the case. This first graph shows that basically all of the sample, both the wild type and the full construct, showed the expression of GFP, which was problematic.

This second graph shows just the wild types with different concentrations of Sodium. The data shows that all of the samples hold steadily at a range of 170- 210 Arb. Units, but then it seems to show some expression of GFP at around the 11.5 hour and 15 hour mark. If the Absorbance was just kept constant at the 170-210 range without any changes or spikes, the data would conclusively prove that our construct was functional, but that was not the case.

This third graph shows the 400mM NaCl with the wild type and the full construct. When we looked at the data as a whole, there seemed to be no discernable proof that our construct was working, but when we isolated the data and looked at only the 400mM NaCl data, there seemed to be some positive data. As you can see the lower line A8 is the wild type, which is lower than all the rest of the lines, which could prove that our construct is somewhat functional. The issue with our data is that only a particular set of data seemed to show positive results. With more time, our team would have been able to replicate the experiment to see if the data collected from the 400mM samples could be replicated; however, our team was only able to run our sample just once prior to the end of our internship.

Conclusion

The inconclusive data we believe could be a result of 2 things. 1st it could be an issue with the construct itself. Since we were having problems getting/finding correct cut sites for the full construct and the fact that pgaA was giving us quite the headache, it could be possible that the colonies were either duds or only contained the partial construct. The second reason could be due to the unknown concentration of NaCl that would trigger the response of this construct. Due to the limited study in this field, we were unsure as to how much sodium to introduce to the testing. Therefore, it could be possible that we were either over or under the threshold for there to be any reliable/positive results.

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