Difference between revisions of "Part:BBa K515100"

Experimental Data

Salkowski Assay

Figure 2: Standard curve of Salkowski assay made with synthetic IAA in LB

Figure 3: Cuvettes used to measure OD for the standard curve. As IAA concentration increases, the solution turns red.

The Salkowski assay is a colourimetric assay that detects IAA with high specificity among other indoles. There are many different types of Salkowski reagents which work at different concentration ranges of IAA and with varying specificity. They all vary slightly in composition and measurement method. We used the most specific reagent according to a paper which works at a concentration range of 0-260 µM. Modelling of the IAA producing construct informed us that IAA production would be within this range. This standard assay is the simplest way to determine whether there is IAA present in solution. First, we created a standard curve with increasing IAA concentration in LB broth using synthetic IAA (Figure 1&2). This was used to determine IAA concentration from OD measurements of IAA-producing E. coli DH5α.

Figure 4: Results from trial 1 of Salkowski assay with cell filtrate of IAA-producing E. coli DH5α. Filtered through a 0.2 µm pore filter

Figure 5: Visual results correlating with OD measurements. The eppendorf on the right contains IAA producing E. coli DH5α and the eppendorf on the left contains control E. coli DH5α.

We found that our IAA producing E. coli were producing approximately 55 µM IAA. From modelling, we have determined that our construct would be able to produce 72.25 μM IAA, which shows that we were in the correct order of magnitude. E. coli are known to naturally express IAA, although the pathway is uncharacterised, which is why all of our controls showed moderate levels of IAA production^[3]. However, cells containing the Auxin Xpress construct have repeatedly shown to produce almost twice as much IAA.

IAA is known to degrade quite rapidly so we tested the effect of light exposure on IAA detection by Salkowski. Interestingly, from testing the Salkowski assay on synthetic IAA in LB left overnight in dark versus light suggests that light exposure does lead to IAA degradation (Figure 6).

Figure 6: Testing the effect of light exposure on synthetic IAA stability. The cuvette on the left shows the colour change at point zero. The three middle cuvettes were left in the dark overnight and the three on the right were left exposed to light, after which Salkowski reagent was added to all samples to observe colour change.

Figure 7: Salkowski assay performed on IAA producing E. coli and control E. coli incubated for 20 hours in different media. All samples were incubated in the dark.

Due to the results of the light exposure test, all future cultures were incubated in the dark. We did another assay on E. coli DH5α cultures expressing the auxin construct to compare IAA production when incubated in two different media, LB and tryptone broth (Figure 7). Surprisingly, the results suggest that IAA production was optimal in LB, although the OD at 600 nm of cultures grown in tryptone broth (very nutrient rich) was much higher. We cannot draw a conclusion from this data, however it seems that the IAA producing pathway endogenous to E. coli is much more complicated than anticipated. We may postulate that IAA is not produced when growth conditions are very favourable and cell density is high.

HPLC

Since the BL21 strain of E. coli seemed to be saturating the Salkowski reagent we looked for alternative quantification methods to measure the amount of IAA being produced by BL21 cells containing the Auxin Xpress construct. The extraction method resulted in a large loss in IAA from the cell filtrate and no peaks were detected by HPLC, therefore we ran filtered cell supernatant directly through the column and the peak characteristic to IAA at 227 nm was visible.

From the Salkowski results, we assumed that cells containing the Auxin Xpress construct were producing about 50 uM IAA and therefore the positive control used for HPLC was at the same concentration. From the positive control, only one peak at 227 nm showed up at about 15 mins so we knew that this corresponded to IAA and looked for the same peak in our samples between 15 and 16 minutes.

Considerably smaller IAA peaks were present in the sample and negative control and a difference in magnitude between the two could not be determined. Therefore we needed an even more sensitive approach to accurately determine how much IAA our engineered bacteria were producing.

Figure X: HPLC peak corresponding to IAA. Positive control is 50 uM IAA in acetonitrile. Auxin Xpress is filtered supernatant of E. coli BL21 transformed with the Auxin Xpress construct. Negative control is filtered supernatant of E. coli BL21 transformed with a vector containing only a promoter and antibiotic resistance.

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LCMS

The third test we did to determine the concentration of IAA secreted by our engineered bacteria was liquid chromatography mass spectrometry. This highly quantitative method combines HPLC and mass spectrometry making nano-molar concentrations detectable. Luckily Dr Colin Turnbull and his team were running IAA samples on LCMS and generously ran our samples for us. E. coli strain BL21 were used to maximise IAA output.

The results show that E. coli transformed with our Auxin Xpress construct produce more IAA than cells without the construct (Fig. 9). From nanograms of IAA detected in each sample, we calculated the molar concentration of IAA based on the fact that positive control 1 was LB doped with 50 µM IAA. The post extraction concentrations were nearly 12 fold lower than pre-extraction, however the relative levels of IAA are highly informative. Positive control 2 had much more IAA than positive control 1 because it contained negative control cell culture spiked with 50 µM IAA. We know that E. coli naturally produce IAA which is why the negative control had IAA and why positive control 2 had more IAA than positive control 1.

From this data we know that E. coli engineered with the Auxin Xpress construct, produce 37 percent more IAA than negative control E. coli.

Figure 9: LCMS results show that cells containing the Auxin Xpress construct produce substantially more IAA than control E. coli. Positive control 1 was LB spiked with 50 µM IAA, positive control 2 was the negative control spiked with 50 µM IAA, and the negative control was E. coli BL21 transformed with a vector only containing a promoter and antibiotic resistance.

Figure 10: Peaks produced from LCMS, the peak area of the MRM transition 176-130 was used to quantify IAA. The bottom window shows the peak for authentic IAA run in the same batch compared to the test sample extracted from our engineered bacteria. The peak around four and a half minutes corresponds to another unkown metabolite. The peak at about six and a half minutes corresponds to IAA.

From this data it seems that the IAA levels determined from the Salkowski assay are not very accurate, which is most likely due to cross-reactivity with other indoles. We found our engineered bacteria produced about 50 µM IAA from Salkowski, however LCMS only detects about 28 µM IAA.

References

[1] Spaepen S. et al. (2007) Indole-3-acetic acid in microbial and microorganism-plant signaling. Federation of European Microbiological Societies Microbiology Reviews 31: 425–448.

[2] Palm CJ et al. (1989) Cotranscription of genes encoding indoleacetic acid production in Pseudomonas syringae subsp. savastanoi. Journal of Bacteriology 171(2): 1002-1009.

[3] Ball, E(1938) Heteroauxin and the growth of Escherichia coli. Journal of Bacteriology 36(5):. 559-565.