Part:BBa_K2955003

glpA+glpB

This part combines the two genes related to glyphosate resistance and degradation, glpA and glpB. glpA increases the tolerance of the cell towards glyphosate and has homology with hygromcyin B, a phosphotransferase native to E. coli(Penaloza-Vazquez et al, 1995). glpB was found to be associated with degradation of glyphosate, possibly through the glyphosate oxidoreductase pathway in which the molecule's C-N bond is broken to form aminomethylphosphonic acid (AMPA) and glyoxylate (Penaloza-Vazquez et al, 1995). These two parts were combined into a single plasmid with the ultimate goal of being able to increase the tolerance of the cell towards glyphosate and introduce a means for the cell to degrade the molecule.

Usage and Biology

In order to gauge Roundown’s response to both pure glyphosate and commercial formulations of glyphosate-based herbicides, we measured its growth over time relative to control plasmid carrying cell line. We attempted to induce individual expression of each gene using the promoters pLac and pTet but ran into issues with inhibiting the pTet promoted gene. This meant that the gene glpA, which is associated with increased glyphosate tolerance, being constitutively expressed. In experiments where IPTG is added, the expression of glpB is induced and both genes are operational.

Methods

This part was tested by running growth curves under various conditions. Growth curve data was collected using a Bioscreener, Bioscreen C. Overnight cultures of the full plasmid, which had both glpA and glpB (BBa_K2955002), and a control culture were grown overnight in tubes of LB+chloramphenicol at 37C and 220 RPM. 3.0 mL of the tested concentrations of pure glyphosate and Roundup were prepared with 3 uL of chloramphenicol. IPTG was added to a final concentration of 0.5 mM for experiments where both genes were expressed. Each concentration was tested with 4 replicates. Each well was inoculated with 3 uL of cells and 300 uL of the stock concentrations. The plates were left in the Bioscreener for 96 hours at 37 C and medium shaking. Measurements were taken every 30 minutes. Since glpB was regulated by pLac, it should have limited expression in the absence of IPTG and should be fully expressed when IPTG is added. glpA is preceded by pTet and is constitutively expressed due to a lack of tetR in the cell strain.

Growth in Roundup

The following figure shows the growth of Rounddown relative to the control when both genes were induced when grown in the commercial herbicide Roundup. Rounddown showed improved growth relative to the control. At 3 mM the control displayed a much longer lag phase than Rounddown and at concentrations greater than 3 it was unable to grow at all. Rounddown reached similar OD600 values for growth conditions up to 4 mM Roundup. In 5 mM Roundup its peak OD was reduced but it still grew better than the control at this condition.

Aloza-Vazquez, A. P., Mena, G. L., Herrera-Estrella, L., & Bailey, A. M. (1995). Cloning and Sequencing of the Genes Involved in Glyphosate Utilization by Pseudomonas pseudomallei. APPL. ENVIRON. MICROBIOL., 61, 6.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 376
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]