Difference between revisions of "Part:BBa K4814006"
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<p>Figure 1 & 2. The image of ATRIP-EGFP after UVB 100 J/m^2 exposure. Both tests showed clusters and aggregation of signal in green channel and red channel. (488 nm excitation)</p> | <p>Figure 1 & 2. The image of ATRIP-EGFP after UVB 100 J/m^2 exposure. Both tests showed clusters and aggregation of signal in green channel and red channel. (488 nm excitation)</p> | ||
| − | <h2>FRET results</h2> | + | <h2>FRET results (G+M)</h2> |
<p>After subjecting the cells to UVB treatment at a dosage of 100 J/m^2, we observed a change in the density of both EGFP and mCherry signals. When excited at 488 nm, we noticed that the EGFP signal became weaker following exposure to UVB. However, in contrast, the red fluorescence emitted by mCherry (with an emission range of 570-620 nm) intensified.</p> | <p>After subjecting the cells to UVB treatment at a dosage of 100 J/m^2, we observed a change in the density of both EGFP and mCherry signals. When excited at 488 nm, we noticed that the EGFP signal became weaker following exposure to UVB. However, in contrast, the red fluorescence emitted by mCherry (with an emission range of 570-620 nm) intensified.</p> | ||
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To handle the non-linear nature of the data, we took the logarithm of the values with a base of 2, which brings the values onto a comparable scale. | To handle the non-linear nature of the data, we took the logarithm of the values with a base of 2, which brings the values onto a comparable scale. | ||
| − | In Figure 4 | + | In Figure 4, the data points in the UV- graph are divided into two groups, with a separation occurring at 0.1. We set this value as the cutoff point, indicating the presence of FRET when the data point exceeds 0.1. |
In the UV+ graph, there is a noticeable distinction in the proportion of data points indicating FRET. | In the UV+ graph, there is a noticeable distinction in the proportion of data points indicating FRET. | ||
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| − | + | <img src="https://static.igem.wiki/teams/4814/wiki/lab/human/log2-red-green-ratio-distribution.png" style="width: 600px;"> | |
| − | + | <center><p>Figure 4. Distribution of the Log base 2 Red/Green data before and after UV.</p></center> | |
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Figure 4 & 5. The graph of Log_2 (Red/Green Ratio) distribution before and after UV. | Figure 4 & 5. The graph of Log_2 (Red/Green Ratio) distribution before and after UV. | ||
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To assess the significance of the relationship between the two categorical variables, we employed Fisher's exact test. This statistical test is suitable when dealing with small cell counts. When the two-sided p-value is less than 0.05, it suggests a significant association between the two groups. (MedCalc Software Ltd. Fisher, 2023) | To assess the significance of the relationship between the two categorical variables, we employed Fisher's exact test. This statistical test is suitable when dealing with small cell counts. When the two-sided p-value is less than 0.05, it suggests a significant association between the two groups. (MedCalc Software Ltd. Fisher, 2023) | ||
| − | The result of Fisher's exact test revealed a strong significance between the | + | The result of Fisher's exact test revealed a strong significance between the UV- and UV+ groups (p-value = 0.00122178, p-value < 0.05), indicating a stastical significance. |
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| − | Figure | + | Figure 5. The Mean Log base 2 of Red over Green ratio with standard error before and after UV. Technical sample number = 4 with about 30 data points in each sample. |
References: | References: | ||
Revision as of 15:26, 11 October 2023
ATRIP-EGFP
- NOTE: This part is used together with part BBa_K4814007 (RPA1-mCherry) as a FRET pair.
FRET is using fluorescent proteins as probes to detect the interaction of targeted proteins. The distance-dependent process transfers energy from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor) through intermolecular long-range dipole–dipole coupling once the desired proteins bind (Sekar, R. B. and Periasamy, A., 2003). The critical Förster radius (typically 3-6 nm) at angstrom distances (10–100 Å) can be calculated to increase the accuracy and ensure precise energy transfer. (Alan Mulllan, n.d.) By using FRET, we can therefore observe the interaction of two proteins by measuring the lifetime of the fluorescent proteins attached to them.
As the aim of this design is to detect DNA damages in mammalian cells, we have used CMV promoter and the Lenti virus vector. Please refer to BBa_K4814004 and BBa_K4814005 (ATRIP and RPA1) for detailed explanation of the two proteins involved in the DNA damage checkpoint process.
The EGFP is derived from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC146266/ (same as BBa_K1875003), a mammalian codon optimized enhanced GFP.
Aggregation after UV treatment (Only ATRIP-EGFP)
After exposing the cells to a UVB dosage of 100 J/m^2, we observed aggregation of the EGFP signal (Fig. 1 and 2). Interestingly, fluorescence was detected in both the Green and Red channels. It is important to note that the emission of GFP is dependent on its fluorescence spectra, as mentioned in studies by Sattarzadeh, A. et al. (2015) and Licea-Rodriguez, J. (2019). This fluorescence could potentially be attributed to GFP emitting at around 560 nm.
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