Team:Austin UTexas/Improve
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While being able to measure the metabolic burden of genetic devices is useful in and of itself, it is also important to be able to apply this information towards practical purposes. We sought to determine if the evolutionary stability of a burdensome genetic part could be improved by making changes to its DNA sequence.
The SYFP2 Plasmid
SYFP2 (Part Name BBa_K864100) is a bright yellow fluorescent protein that is closely related to GFP. The 2015 Austin_UTexas iGEM Team found that expression of SYFP2 in cell culture decreased dramatically over time, with cells losing fluorescence in one to three days. They found this was primarily due to the presence of IS10 insertion elements which resulted in loss of function mutations. These IS elements appeared to show preference for a particular hotspot within the sequence of the SYFP2 gene.
Redesign and Measurement
The sequence of SYFP2 was altered to remove this IS10 hotspot (Part name BBa_K3174008). This new part was then cloned and characterized by Kelsey Hu. The 2019 Austin_UTexas iGEM team decided to compare the mutation rate of the new sequence to the original in order to determine if there was a functional improvement in evolutionary stability. Over the summer, Anna Bardenhagen performed experiments in order to determine if the new SYFP2 plasmid was expressed for a longer period of time than the original. She replicated the 2015 Austin_UTexas iGEM team's propagation procedure using TOP10 competent cells transformed with SYFP2 using either the original or redesigned sequence. Using flow cytometry, she measured the fluorescence of propagated cultures over several days and determined that the redesigned SYFP2 gene was expressed for longer than the original sequence.
The following data was collected from trials run during the same period. Figure 1 (on the left) shows that SYFP2 expression from the original sequence is lost within a day of growth. Figure 2 (on the right) shows that SYFP2 expression from the redesigned sequence lasts up to four days.
Figure 1: Fluorescence histograms for TOP10 cells with the original SYFP2 plasmid. The x-axis shows the intensity of fluorescence on a logarithmic scale, and the y-axis displays the number of cells. Each line represents a different day of propagation, and the shaded gray curve is a blank. Peaks to the right of the blank represent fluorescent cells while peaks on or to the left of the blank represent cells that have ceased to be fluorescent. All replicates were grown under the same conditions.
Figure 2: Fluorescence histograms for TOP10 cells with the redesigned SYFP2 plasmid. The x-axis shows the intensity of fluorescence on a logarithmic scale, and the y-axis displays the number of cells. Each line represents a different day of propagation, and the shaded gray curve is a blank. Peaks to the right of the blank represent fluorescent cells while peaks on or to the left of the blank represent cells that have ceased to be fluorescent. All replicates were grown under the same conditions. No data could be collected for Day 3 of Replicate 4.
These figures show that the redesigned sequence is more evolutionarily stable as it takes nearly three more days to break than the original. While removing the IS10 hotspot didn't reduce the metabolic burden of SYFP2, it showed that removing sources of mutations that exacerbate loss of function can increase the longevity of genetic devices.