Revision as of 17:57, 21 October 2019

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.

[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

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, is 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 (BBa_K2955001) 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 30 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.

Results

Growth in Glyphosate

The following graph shows the relative growth of Rounddown when grown in pure glyphosate. While increasing the concentration of glyphosate had limited effect on the peak OD600 reached by the cells, it did extend the lag phase of Rounddown and the control for all increases in concentration. Rounddown was able to reach a similar peak OD for concentrations up to 13 mM over the time period the samples were measured. At 13 mM Rounddown reached a peak OD of around 1.2, while the control only reached an OD of 0.4. It is also interesting to note that the peak OD reached by Rounddown was greater for samples containing glyphosate than the samples grown in just LB. This may be due to utilization of glyphosate as a phosphate source.

                                                            Figure 1

Narrowing in on a concentration of 10 mM glyphosate, it can be seen in Figure 2 that both the control and Rounddown performed better when IPTG was added, with both reaching a higher OD600. Rounddown showed a shorter lag phase, however, a trend that persisted as concentration of glyphosate was increased.

                                                            Figure 2

Around a concentration of 13 mM, Rounddown began to show a clear advantage over the control as shown in Figure 3. The growth of the control was inhibited and its peak OD was at 0.3. Rounddown had a more significant lag phase compared to when grown at 10 mM, but reached a similar peak OD of 1.1. Fully induced Rounddown also grew much better than Rounddown and the control when IPTG was absent. This would indicate that glpB is important to decreasing lag phase time.

                                                           Figure 3                     

Growth in Roundup

The next 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.

                                                            Figure 4

When grown in Roundup, differences in growth between the control and Rounddown start appearing at 3 mM, as shown in Figure 5. The control in this condition showed no growth in the absence of IPTG and had a highly extended lag phase when grown in 0.5 mM IPTG. In contrast, Rounddown grew in the absence of IPTG and adding IPTG further improved growth. This indicates that glpA on its own increases the tolerance of the cells towards Roundup and that glpB further improves its ability to grow under these conditions.

                                                             Figure 5

Figure 6 is similar to Figure 5, but at a growth condition at 4 mM. At this concentration the control’s growth is totally inhibited at all conditions. Rounddown reaches lower peak OD at this concentration than at 3 mM, but the overall trend of IPTG induction of glpB improving growth is still seen.

                                                              Figure 6

Sequence and Features