Part:BBa_K2955001

glpA

glpA is a gene native to the bacterium Burkholderia psuedomallei (formerly Psuedomonas) that was found to confer resistance to glyphosate by Penaloza-Vazquez et al (1995). Glyphosate is the main active ingredient in many commercial herbicides. This gene is the first in our two gene system we consucted with the ultimate goal being the degradation of glyphosate. This gene was meant to increase the survivability of E. coli in the presence of glyphosate, based on the results reported by Penaloza-Vazquez et al (1994). The exact mechanism by which resistance to the molecule is increased isn't known, however. Expression of this gene was regulated by pTet.

[1]Penaloza-Vazquez, A., 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

Usage and Biology

glpA action is believed to be associated with the transport of glyphosate across the cell membrane due to it's homology with native E. coli phosphotransferases. In order to gauge this gene's impact on the ability of the cell to survive the presence of glyphosate, it was tested in both pure glyphosate as well as a commercial formulation of the herbicide, Roundup. Growth curve data was used to determine if glpA confers a survival advantage the cells compared to other E. coli without the gene. The promoter for this gene was pTet but as the cell line used lacked tetR, glpA was constituvely expressed.

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. 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 meadium shaking. Measurements were taken every 30 minutes. In order to test glpA independently of glpB, no IPTG was added to the wells. Since glpB was regulated by pLac, it should have limited expression in the absence of IPTG.

Results

When grown in pure glyphosate, the construct Rounddown showed very little difference in growth compared to the control when only glpA was expressed. This can be seen in the following figure, which displays the growth curves obtained when Rounddown and the control were grown in pure glyphosate with no IPTG. Peak OD600 decreased for both cell types with each increase in concentration.

When grown in Roundup, a commercial formulation containing surfactants in addition to glyphosate, the presence of glpA had a profound effect. As shown in the following figure, the presence of glpA allowed cells to survive at concentrations up to 4 mM in Roundup, while cells that did not contain the gene were unable to grow past 2 mM concentrations of Roundup. The control showed a very long lag phase when grown in 3 mM, and growth was just starting to increase as the end of the measurement period occurred. The peak OD600s for Rounddown at concentrations of 2 mM, 3 mM, and 4 mM were all very similar, though they were moderately lower than Rounddown grown in the absence of herbicide.

While it is unexpected that there is no difference in growth seen between Rounddown and the control in pure glyphosate, there is a very clear advantage for Rounddown when grown in Roundup. Roundup in general is more toxic, as growth inhibition is seen at much lower concentrations of glyphosate than when grown in a solution containing a pure form of the molecule. This would indicate that the surfactant or some part of the formulation is negatively impacting growth and that glpA helps the cell to survive in the presence of these factors as well as in the presence of glyphosate. In terms of applications in bioremediation, it is preferable that the gene provides an advantage for a formulation of the herbicide, since all large scale herbicidal applications use formulated products.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]