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Proof of Concept and HeLa Positive Control

We cannot determine at this time whether our p14ARF expression sensor functions in its intended environment because we were unable to perform a successful transfection into HeLa. In addition to a mock transfection and an untransfected well to act as negative controls, we performed a transfection of pComet, a well-documented plasmid known to produce fluorescence in mammalian cells, as a positive control. In each of our two transfections, neither the HeLa cells transfected with our sensor nor the positive control expressed GFP under a fluorescent microscope. We took this as evidence that the transfection itself failed, not just our sensor, although this does not rule out the possibility that our sensor did fail.

As we grew our cells, we experienced difficulties keeping them alive. On the fourth week, we noticed that the caps of the flasks used to store cells were fastened tightly, preventing proper airflow. If this was done more than once during the four weeks, then it may explain our cells’ low fitness. Cells were also not passaged enough during Thanksgiving break.

Given the fact that we took extreme care to follow procedures exactly and always work in pairs to verify that the person in the hood was adding the right amounts at the right times, we believe it is unlikely that the poor results were caused by experimental error. We believed the first trial failed because our cells were too confluent (approximately 80%), preventing healthy division, and because we performed a transfection of suspended cells, which has reduced success. While our cells in the second trial had a healthy confluence of 60% and had been allowed to attach to the well plate, by this time they were four weeks old and likely too unhealthy because of the lack of airflow to take up a genetic construct.

With these results, we cannot provide evidence to demonstrate whether our sensor functions in its intended environment. More testing is needed to provide any proof or disproof of concept.

Part 2: We performed a successful double transformation of the E2F1 plasmid into S. cerevisiae containing the p14ARF construct. Yeast with both constructs survived on uracil leucine selective media, but not untransformed cells, which suggests no contamination. Figure 1 shows one of our successful double transformation plates.

Figure 1: Successful double transformation of p14ARF and E2F1 plasmids into S. cerevisiae.

Before measuring the fluorescence we grew up cells in fructose-based leucine DO media with 2% galactose for one group, 3% galactose for another, and glucose-based uracil leucine DO media for a negative control group.

We measured the fluorescence of the double transformed cells at Excitation 500 and Emission 531 as well as Excitation 510 = nm and Emission 540 = nm with normal yeast as an additional negative control and reference for normal fluorescence levels. For double transformed cells grown in 2% galactose solution, the average normalized fluorescence levels corrected for concentration at OD600 and the blank readings were 147.10 at Excitation = 510 nm and Emission = 540 nm. For double transformed cells in 3% galactose, the average normalized fluorescence levels were 160.75. For double transformed cells in glucose-based media (which should have repressed the actuator), the average normalized fluorescence levels were 466.86. For normal cells, the average normalized fluorescence levels were 172.09.

From this data, we may conclude that double transformed cells failed to produce meaningful fluorescence compared to that of our control groups. Because our positive assay of the sensor construct in HeLa cells produced no results, we have no indication whatsoever whether it was our sensor that failed, our actuator, or both.

Assuming E2F1 was properly expressed by the actuator, the sensor could have failed to express GFP for many reasons. We believe the most likely cause of failure to be incompatibility between the sensor and S. cerevisiae’s transcription-translation machinery. Although yeast contains CpG island promoters, even if E2F1 binds to its cognate binding sites within the enhancer and promoter, it is not clear how it will activate yeast RNAP and transcription. Furthermore, assuming transcription occurred properly, there is no known yeast ribosome binding site for the mRNA, although our sensor contains similar enough sequences to some yeast RBSs along with a kozak site. Since we lack results from our positive control to suggest otherwise, another likely cause for failure could be the lack of separation between the enhancer and actuator. As we mentioned in our introduction, very short distances make it difficult to form a loop structure that brings the enhancer and promoter into contact due to the rigidity of DNA. It is possible that this failure to form a loop prevented E2F1 from contacting the promoter after it bound to our enhancer. Finally, some unknown mutation could have occurred on the vector, rendering it nonfunctional, but this is the least probable explanation.

Assuming that it was our actuator that failed, a number of factors could have prevented proper expression of E2F1. In this case, we believe the most likely cause of failure to be E2F1 stability. Research demonstrates that E2F1 is unstable in stages of the cell cycle besides G1 exit and S phase, and its stability must be regulated dynamically throughout the cell cycle [1]. Without these regulatory elements present in human cells, it is likely that E2F1’s instability in yeast prevented proper expression. Other possibilities remain. Though our research demonstrated that yeast does not contain proteins homologous to the mammalian E2F family, it does contain a protein SBF that performs the same function [2]. E2F1 could have bound with some element in yeast with affinity for SBF that prevented detection by the sensor. Furthermore, a completely unknown molecule in yeast could have also inhibited, interfered with, or degraded E2F1. Besides this, the mRNA secondary structure might not be able to interact with yeast machinery, or lack of introns could have prevented successful mRNA maturation [3]. The conditions within yeast, including temperature, pH, solute concentrations, may not have been suited for producing this mammalian protein. Unfortunately, we cannot say this definitively because the ideal conditions for E2F1 synthesis have not been characterized. Finally, some unknown mutation could have occurred on the vector, but this is the least probable explanation.

Negative Control

We performed a successful transformation of the p14ARF sensor into S. cerevisiae. Yeast with the construct survived on leucine selective media, but not untransformed cells or cells transformed with the E2F1 actuator, which suggests no contamination.

We measured the fluorescence of the transformed cells in PBS using a plate reader set to Excitation 500 and Emission 531 as well as Excitation 510 and Emission 540 with normal yeast as a negative control and reference for normal fluorescence levels. For the transformed cells, the average normalized fluorescence levels corrected for concentration at OD600 and the blank readings were -27.76. For the normal cells, the average normalized fluorescence levels were 172.09. Although our data demonstrates that the sensor alone does not express fluorescence within yeast. Therefore, there is no element within yeast, identical/homologous to human oncogenes or otherwise, that activates GFP expression in our p14ARF sensor. However, without data from our positive control and negative data from our experimental group, we cannot say whether this is due to our sensor successfully activating only upon receiving desired inputs or our sensor failing to activate at all. At this stage, all we know is that the sensor does not produce false positives.

Figure 2: Results of fluorescent plate reader assay at Excitation 510 nm and Emission 540 nm before normalization. Well number is indicated on the left, and the contents of the well is indicated across the top. The top table shows the measured fluorescence at wavelengths 510 and 540, while the bottom table shows the OD 600 values.

Figure 3: Results of Plate Reader Assay at Excitation 510 nm and Emission 540 nm with normalized values. The fluorescence intensity indicated was calculated relative to control values. Afterwards, the fluorescence intensity was normalized by the OD 600 values of each respective yeast cell culture. Well number is shown in the left column and well contents is shown across the top of the table.

Figure 4: Figure 4: Fluorescent intensity of double-transformed cells induced with 3% or 2% galactose, in glucose based (Gal1 repressive) media, and as compared to wild-type yeast at Excitation = 510 nm and Emission = 540 nm. 2% and 3% Galactose were added to wells to induce the production of the actuator E2F1. Glucose-based media served as the negative control. The fluorescent intensity indicated was calculated relative to control values. Afterwards, the fluorescent intensity was normalized by the OD 600 values of each respective yeast cell culture.

Figure 5: Negative control (wild type yeast and p14ARF single plasmid yeast) at Excitation 510 nm and Emission 540 nm. The p14ARF single plasmid yeast is shown to not produce false positives, with low fluorescence values. The fluorescence intensity indicated was calculated relative to control values. Afterwards, the fluorescence intensity was then normalized by the OD 600 values of each respective yeast cell culture.

Sources: 1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC15859/ 2. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2776103/ 3. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3325483/

Additional Source: https://www.neb.com/tools-and-resources/feature-articles/bypassing-common-obstacles-in-protein-expression

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