Team:Northwestern/Results
RESULTS
DETERMINING EXPOSURE METHOD
RESULTS
After exposing 20ng of our synthesized DNA with our UVB transilluminator @ 304nm and 8 watts/cm2, as well as our UVC hand lamp @ 254nm and 11.9 watts/cm2 for 10 minutes, we found that the majority of strands exposed with the UVC hand lamp remained as a single fragment, illustrating that dimers were, in fact, present in the restriction sites of the strand (Fig. 3). We also found that the UVC hand lamp was much better at damaging the DNA than the UVB transilluminator for two possible reasons - 1) the UVC light shines with higher energy, or 2) the petri dish used to hold the DNA samples absorbed UV radiation from the transilluminator. Unlike the UVB transilluminator, the UVC hand lamp provides direct exposure to the samples with no barriers. If we had the resources to acquire a UVB hand lamp, we could’ve developed a control to establish which factor had the greater impact on the amount of DNA that was cut versus uncut between the two exposure methods. These findings establish that our UVC hand lamp was capable of inducing thymine dimers, prompting us to use it to expose our cells in our final in vivo assay.
Figure 1. Exposed and unexposed strands electrophoresed on a 2% agarose gel with Hi-Lo ladder. Classifications of each lane can be found in Table 2.
Table 1. Labeling for the lanes of Figure 1
1
Hi-Lo Ladder
2
Unannealed Top strand used as a control for the effect of the annealing process on gel electrophoresis
3
Unannealed bottom strand used as a control for the effect of the annealing process on gel electrophoresis
4
Unexposed and EcoRI digested strand
5
UVB exposed and EcoRI digested strand
6
UVB exposed through plastic seran wrap and EcoRI digested, as a control for the amount of UV absorbed by the plastic petri dish
7
UVC exposed and EcoRI digested
8
UVC exposed with petri dish lid and EcoRI digested
| 1 | Hi-Lo Ladder |
| 2 | Unannealed Top strand used as a control for the effect of the annealing process on gel electrophoresis |
| 3 | Unannealed bottom strand used as a control for the effect of the annealing process on gel electrophoresis |
| 4 | Unexposed and EcoRI digested strand |
| 5 | UVB exposed and EcoRI digested strand |
| 6 | UVB exposed through plastic seran wrap and EcoRI digested, as a control for the amount of UV absorbed by the plastic petri dish |
| 7 | UVC exposed and EcoRI digested |
| 8 | UVC exposed with petri dish lid and EcoRI digested |
UV SENSITIVITY VALIDATION
Our results suggest that there is no significant difference in induction at any of the UV doses for our part BBa_K3269001 (Fig.2). However, we ran our assay in tandem with our improve a part assay, in which cells with part BBa_K079050 demonstrated a significant difference in fluorescence standardized over OD. From this result, we can conclude that the problem is not with our overall procedures, but with either the UV dose given to our cells or the design of our part. Additionally, our results from the improve a part assay (Fig. 3) show an unexpected pattern where there is no difference in initial induction, but instead a difference in degradation rates of sfGFP. One possible hypothesis for this trend could be that part BBa_K079050 could remain turned on for a longer duration after increasing UV doses, leading to a less steep decline in Fluorescence/OD. In this way, the part still provides a distinction between UV dose, but not in the manner it was originally designed for.
Figure 2. A graph of Fluorescence standardized over OD600 of our part (BBa_K3269001), referred to here as UvrA, versus another part of our own design that constitutively expresses sfGFP (BBa_K3269002), referred to as Const. The time next to each title is the duration of UV exposure prior to being placed into the plate reader and directly corresponds with the UV dose.
Figure 3. Fluorescence standardized with OD600 over 3 hours of the part we intended to improve BBa_K079050, referred to here as LexA2, and our genetically altered part: BBa_K3269004, referred to here as LexA1. Discussion of these results can be found in the previous paragraph.
This unexpected trend, as well as the inability of our part to become induced, could be the result of exposing the cells to the UV transiently, instead of continuously while taking fluorescence measurements. Conceptually, when UV is exposed transiently, only the first generation of cells would have their genomes damaged by the UV, where they halt their replication process until they repair that damage. Therefore, the following generation would not have any increased promoter activity, as they had not received any of the initial UV doses. Continuous exposure to UV would provide consistent experimental conditions from generation to generation, possibly resulting in proper induction. When compared to the LexA2 part, however, it may be that the design of our plasmid may not have recruited transcription and expression mechanisms enough to activate our part.
Figure 2. A graph of Fluorescence standardized over OD600 of our part (BBa_K3269001), referred to here as UvrA, versus another part of our own design that constitutively expresses sfGFP (BBa_K3269002), referred to as Const. The time next to each title is the duration of UV exposure prior to being placed into the plate reader and directly corresponds with the UV dose.
Figure 3. Fluorescence standardized with OD600 over 3 hours of the part we intended to improve BBa_K079050, referred to here as LexA2, and our genetically altered part: BBa_K3269004, referred to here as LexA1. Discussion of these results can be found in the previous paragraph.