Difference between revisions of "Team:Valencia UPV/Contribution"

An agroinfiltration and subsequent luciferase assay were performed in order to study gene expression at transcriptional level. The final Optical Density of reporter constructs (Figure 2a and Figure 2c) was 0,02 and the optical density of PhiC31 construct (Figure 2b) was 0,01. A triplicate sampling of different plants was performed in order to take account for biological variability due to unknown or uncontrollable conditions.

Register assembly constructs (Fig. 2a and 2b) and the controls (Fig. 2e and 5f) were agroinfiltrated and after 54h post-infiltration, leaves were sampled. After 48h, all leaves were also sampled. Overall, two points of samples were taken in the assay.

After analyzing the data obtained from luciferase assay, it can be observed at Figure 2:

1. A significantly difference between the OFF and the ON state expression levels.
2. The recombinase expression under a weak promoter (pNOS promoter) shows the same luciferase expression that the constitutive positive control (genetic construct which is expressing constitutively luciferase protein under the strong promoter).
3. However, the recombinase expression under the strong promoter shows the same expression level of the constitutive weak control (genetic construct based on the constitutive expression of luciferase under a pNOS weak promoter)

The likelihood of producing the inversion event should be directly proportional to the luciferase protein concentration inside plant cell. Consequently, more quantity of phiC31 recombinase entails more probability of activating reporter gene expression. However, this experiment shows that once recombinase expression exceeds a certain threshold, the effect changes so the likelihood of up-regulating the reporter gene expression decreases.

In order to provide robust evidences to the hypothesis explained above, a subsequent experiment (https://2017.igem.org/Team:Valencia_UPV/Experiments#Phic31Per) was performed. In this case, PhiC31 behavior was analyzed while being regulated by a weak promoter (pNOS) together with an optical density (A.tumefaciens culture concentration) was 10-fold higher than the used in aforementioned experiment (Figure 3).

In order to provide robust evidences to the hypothesis explained above, a subsequent experiment (https://2017.igem.org/Team:Valencia_UPV/Experiments#Phic31Per) was performed. In this case, PhiC31 behavior was analyzed while being regulated by a weak promoter (pNOS) together with an optical density (A.tumefaciens culture concentration) was 10-fold higher than the used in aforementioned experiment (Figure 3).

Figure 3. a) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 attachment sites (attB and attP). It represents the negative control of our experiment. Only when ΦC31 inversion occurs, luciferase protein will be expressed. b) Genetic construct that allows constitutive expression of phiC31 in the plant under a strong promoter c) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 recombined attachment sites (attR and attL). In normal basis, the promoter is inverted, allowing the expression of luciferase protein. It represents the positive control of our experiment. d) Plot representing PhiC31 behavior when infiltrated at OD 0,1.

Conclusion: It was demonstrated the proper phiC31 functioning and determined its behavior under different protein concentrations and promoter strengths. Although the expected hypothesis may be that the more recombinase concentration the more inversion events, the obtained results suggest the existence of a threshold above which that assumption is not true. Therefore, a low recombinase expression is needed in order to maximize recombinase action.

Our team has improved the characterization of the Phytobricks (PhiC31) documenting our experimental design and the results that were obtained. All data is also documented on the Main Page in the Registry.