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Comparing Genetic Device Stability 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. 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 24 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 Top10 strain is quickly breaking down, while the MDS strain has remained stable. After four days, the Top10 group of SYFP appeared to have a population that was mostly none fluorescent (See Figure 2). On the sixth day, all of the MDS42 cultures, the third and fifth BL-21 cultures, and the second BW-25113 culture were carried forward and recorded using a flow cytometer.

In the summer, we explored fluorescent stability and mutational variance in different strains of E. coli. In particular, since MDS-42 is a strain of E. coli without transposons, in that strain we hoped to observe mutants that otherwise would have been outcompeted* by mutants with the insertable elements.

Procedure

To continue studying evolutionary stability, we began a variation of our spring experiment. Instead of studying only Top10 E. coli, we expanded our experiment to include other common strains, specifically: BL-21 (DE3), BW-25113, and MDS-42. The MDS-42 cells had the added benefit of containing only a minimal genome.

Before propagating the cell cultures, for each strain, we streaked transformed cells from frozen stocks on to an LB/CAM plate and let them incubate overnight. The next day, we chose six indepedent and fluorescent colonies for each strain. To ensure all cells were retrieved for a more accurate generation time, we pierced the full depth of the agar with a micropipettor tip large enough to encompass the entire colony. We then placed the colony in 10 mL of LB/CAM media and grew all 24 cultures overnight in the shaker at 37° C and 215 RPM. We refer to these cultures as 'Day 1' in our results. The next day, we began the propagation/mutation* phase of our experiment.

From this point forward, each morning we retrieved the overnight cultures from the shaker and checked for fluorescence using a blue light, recording any observations. Then, we would vortex each culture to homogenize the liquid, we used 10 μl to start a fresh 10 mL overnight culture with LB/CAM media. Next, we froze 3 mL of culture in 15% glycerol at -80° C for storage and spun down 4.5 mL of culture for minipreps. In the spring, we used a dark reader to determine a single fluorescence value. However, over the summer we switched to using flow cytometry as a more accurate measure of fluorescence. So, with the remaining 2 mL of culture, every few days we would use the flow cytometer to determine the proportion of cells which were still fluorescent.

After a culture appeared to stabilize at either a complete or partial loss of fluorescence, we stopped pushing the culture forward.

Results

Although we are still awaiting sequencing results, data from the flow cytometer helps to elucidate strain to strain differences in stability.

Flow data for the Top10 cultures corroborate findings from the spring--fluorescence diminishes quickly and precipitously. Max fluorescent cell intensity typically centers around 10^6, while our control media (with zero fluorescence) centers around 10^4. By the second day, the fluorescence cell populations were almost completely absent and on the fourth day, we stopped pushing Top10 forward.

BW-25113 behaved similarly, on the second day we consider lessthan 20% of the cells to be still fluorescing. However, several of the samples appeared to maintain a small proportion of the cells (20%) with reduced (~4*10^4) fluorescence.

Most BL-21 (DE3) cultures experienced a similar, drastic reduction in fluorescence. However, 2 of the 6 in these strains maintained a small fluorescent population, at approximately 10^5, up to day 3, with one persisting until Day 4.

Finally, fluorescence in MDS-42 cultures endured the longest. Three of the samples had lost fluorescence by Day 3, while the remaining three samples maintained a reduced fluorescence level (~2*10^5) until Day 8.

In summary, Top10 cells were most unstable, with nearly all fluorescence being lost by Day 2. In contrast, MDS-42 cultures exhibted the best stability but also the most variance, with some cultures breaking after only three days and some enduring for an additional five days. BW-25113 and BL-21 (DE3) samples experienced moderate fluorescence stability, with trends less extreme than Top10 or MDS-42.

Figure 1 (TOP10) - Each colored line corresponds to a sample replicate of transformed Top10, while the gray area is a blank. The x-axis represents the intensity of fluorescence on a logarithmic scale while the y-axis represents the number of cells that exhibited a particular fluorescent value. Each blank (grey area) is the fluorescent value of a reading of PBS (Phosphate-buffered saline). Peaks to the left of the end of the blank represent cells that have ceased producing fluorescence proteins while cells past this point have continued production.

Figure 2 (BW-25113) - Each colored line corresponds to a sample replicate of transformed BW-25113, while the gray area is a blank. The x-axis represents the intensity of fluorescence on a logarithmic scale while the y-axis represents the number of cells that exhibited a particular fluorescent value. Each blank (grey area) is the fluorescent value of a reading of PBS (Phosphate-buffered saline). Peaks to the left of the end of the blank represent cells that have ceased producing fluorescence proteins while cells past this point have continued production.

Figure 3 (BL-21 (DE3)) - Each colored line corresponds to a sample replicate of transformed BL-21 (DE3), while the gray area is a blank. The x-axis represents the intensity of fluorescence on a logarithmic scale while the y-axis represents the number of cells that exhibited a particular fluorescent value. Each blank (grey area) is the fluorescent value of a reading of PBS (Phosphate-buffered saline). Peaks to the left of the end of the blank represent cells that have ceased producing fluorescence proteins while cells past this point have continued production.

Figure 4 (MDS-42) Each colored line corresponds to a sample replicate of transformed MDS-42, while the gray area is a blank. The x-axis represents the intensity of fluorescence on a logarithmic scale while the y-axis represents the number of cells that exhibited a particular fluorescent value. Each blank (grey area) is the fluorescent value of a reading of PBS (Phosphate-buffered saline). Peaks to the left of the end of the blank represent cells that have ceased producing fluorescence proteins while cells past this point have continued production.

Discussion

Overall, MDS-42 was best able to maintain fluorescence throughout the duration of the experiment. We speculate that, without the very advantageous mutant that arises from transposons, other mutations were able to compete and resulted in moderate maintenance. These mutations may not have directly affected the SYFP2 coding sequence. For instance, auspicious mutations could have occurred in the genome, or other factors such as the plasmid copy number or antibiotic resistance gene could have been altered. Future analysis of the sequencing data will further illuminate what caused the trends seen in these graphs.