Team:Austin UTexas/Project/Strain Study

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Summer 2015 Data: Fluorescent Proteins in Different Strains


Assessing Yellow Fluorescent Protein genes for stability


Four strains of E. coli were selected to be transformed for the:Top-Ten, MDS-42, BL-21 (DE3), and BW-25113. These Top-Ten, BL-21 (DE3), and BW-25113 were all selected because of the fact that they are commonly used strains in research of synthetic biology. The MDS-42 strain was selected because that strain has many of it’s IS elements removed from its genome. Previous research in the spring has shown that a major cause of mutation in genetic devices is transposable elements inserting into the plasmid. So the hypothesis is that the MDS strain will exhibit greater longevity in terms of fluorescence than the three other strains.

All four strains were transformed with three plasmids that were designed and constructed in spring of 2015. The plasmids contained the same backbone (pSB1C3) and the same vector- a medium promoter and ribosome binding site. The three plasmids differed in only the type of yellow fluorescent protein used: Yellow Fluorescent Protein, Super Yellow Fluorescent Protein, and Enhanced Yellow Fluorescent Protein. These three were selected because of the variance in stability patterns they expressed in the spring semester.
Fig.1 description here
After each strain was transformed with each plasmid, they were grown into over-night culture tubes and then preserved in glycerol stocks.


After some time, the four stocks with the Super-Yellow Fluorescent Protein were grown in media in replicates of six. There were twenty four samples in all. Each culture was grown overnight, a sample was used to carry forward to the next day, a sample was taken for flow cytometry, and glycerol stocks were made. Samples were taken to be analyzed for remaining fluorescence using flow cytometry. Re-suspension of each culture using PBS preceded the use of the flow cytometer. Each sample was then pipetted into a well in a 96-well plate, with every six samples separated by a well filled with PBS only. The flow cytometer reads to fluorescent value of each well by sipping each well automatically using a syringe. The media flows from the syringe and into cytometer to be passed through a laser which counts the number of cells (called events) and the intensity of the fluorescence and repeats this for each filled well. The first three days of samples that were read using flow cytometry are seen in Figure 1. The x-axis is the magnitude of fluorescence, which is using a logarithmic scale. The y-axis is the count of cells or objects in sample with a particular fluorescence. By looking at the counts of positive fluorescence from day to day, it is clear that the Top-Ten strain is quickly breaking down, while the MDS strain has remained stable. After four days, the Top-Ten group of SYFP appeared to have a population that was mostly none fluorescent (SEE Figure 2). On the sixth day, all of the MDS strains, the third and fifth BL-21 strains, and the second BW-25113 strain were carried forward and recorded using the flow cytometer.

Fig.2 PLACEHOLDER FIGURE