High throughput screening of β-glucuronidase (GUS) reporter in transgenic microalgae transformed by Agrobacterium tumefaciens

Abstract

GUS (β-glucuronidase) is a chemiluminescent reporter gene that has been used in E. coli, fungi, yeast, mammalian cells, plant cells and microalgae. Currently, the GUS gene fusion system used for detection of GUS enzyme activity is carried out by cell lysis and requires 500 μl cell lysate. Hence, GUS activity cannot be assessed by high throughput screening (HTS) method. This study addresses this by using a HTS technique to quickly isolate transgenic strains (shown using Chlorella vulgaris) expressing high GUS activity in a 96 well microplate format. In this technique, quantitative results were obtained without carrying out cell lysis and all the experiments were carried out in a 96 well microplate. The method developed is cost effective, less labor intensive and can be carried out in a timely manner.For this, a new GUS reporter vector pBIN + TetR + TetO was developed, followed by transformation (Agrobacterium tumefaciens), screening and characterization of the transgenic C. vulgaris. In the screening study, strain number 18 showed the highest fluorescence intensity (16,988 ± 1168). The GUS enzyme was found to be stable for >8 h for intact cell and lysed cell studies. The optimum concentration of Triton X-100 to release the product (4-Methylumbelliferone) into buffer was 0.1% and the fluorescence intensity was 28,397 ± 787. The values of Km and Vmax of the recombinant GUS for lysed cells were 0.1304 ± 0.0101 mM and 0.35 ± 0.004 pmol 4-MU/min/ml of crude cell lysate respectively.