The purpose of this year’s interlab protocol was to take the absorbance of the bacterial stain E. Coli (created by transforming the plasmid) and standardize the fluorescence rate for future use at iGEM. Our next week of interlab lab work was split into two days: calibration day and cell measurement protocol day. The team quickly learned the importance of accurate pipetting as we had our first experiences with the plate reader.

We followed all the protocols provided by iGem, so click here to see the methods and materials we used.

Results:

After overnight incubation, we put out the samples and counted the one that had less than 300 colonies. After counting the samples, we multiplied with the dilution factor given in interlab to calculate CFU/mL. We had 6 samples each for positive and negative controls, and it was categorized by dilution 3, 4, and 5, and each sample had a different dilution factor according to the protocol. We noticed that there were a samples , so some of the data had to be left blank. CFU stands for colony forming unit, so the number recorded as CFU/mL shows the number of colonies present for every 1 mL of sample (similar as concept of molarity). And the concentration of bacteria is directly related to the fluorescence. If there is more concentration, it will fluorescence more. The measuring of absorbance also helped us to see the concentration, since high concentration of bacteria will scatter the light more than the diluted samples, resulting in the high absorbance value.

In this graph, we analyzed the relationship between absorbance and particle count. We found out that high absorbance means more scattering due to the high concentration of bacteria, and they have linear relationship

This graph compares absorance and the particle count, and we found that slight change in absorbance will change the particle count exponentially

After we analyzed the data of fluorscence during the cailibration process, we found that there is a direct relationship between fluorescein concentration and fluorscence

On this standard curve, we noticed that there is a minimum amount of fluorescein needed to express fluorescence. The non zero y intercept shows that example.

The FilterMax F5 Multi-Mode Microplate Reader we used was courtesy of Dr. Finke at University of Washington Tacoma. Thank you Dr. Finke!