Objective

Testing the performance of phiC31 recombinase using the register assembly construct with the attachment sites PxB.

Plant Chassis

Nicotiana benthamiana

Parts

Figure 1. 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, allowing the characterization of luciferase dynamic. b) Genetic construct that allows constitutive expression of phiC31 in the plant. 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.

Method

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 1a and Figure 1c) was 0,02 and the optical density of PhiC31 construct (Figure 1b) was 0,05. A triplicate sampling of different plants was performed from 36h post infiltration onwards in order to take account for biological variability due to unknown or uncontrollable conditions