Difference between revisions of "Part:BBa K515100"
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| − | <p>We found that our IAA producing <i>E. coli</i> were producing approximately 55 µM IAA. From modelling, we have determined that our construct would be able to produce 72.25 | + | <p>We found that our IAA producing <i>E. coli</i> 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. <i>E. coli</i> are known to naturally express IAA, although the pathway is uncharacterised, which is why all of our controls showed moderate levels of IAA production<sup>[3]</sup>. However, cells containing the Auxin Xpress construct have repeatedly shown to produce almost twice as much IAA. </p> |
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Revision as of 15:30, 21 September 2011
IAA biosynthetic genes under control of the Pveg2 promoter
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 547
Illegal BamHI site found at 1492 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 254
Illegal NgoMIV site found at 2835 - 1000COMPATIBLE WITH RFC[1000]
This BioBrick has been sequence verified.
Background
The IAM pathway is a two step pathway which generates indole-3-acetic acid (IAA), also known as auxin, from the precursor tryptophan. IAA tryptophan monooxygenase (IaaM) BBa_K515000, catalyses the oxidative carboxylation of L-tryptophan to indole-3-acetamide which is hydrolyzed to indole-3-acetic acid and ammonia by indoleacetamide hydrolase (IaaH) BBa_K515001. There are several different pathways that produce indole-3-acetic acid.[1]IaaM and IaaH originate from P. savastanoi and have been expressed in E. coli previously, and shown to secrete auxin into the cell supernatant.[2]
Figure 1: Different pathways can be used to produce IAA. This construct follows the IAM pathway which involves genes IaaM and IaaH to convert tryptophan to IAA via the IAM intermediate.
Experimental Data
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 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 (Fig. 1&2). This was used to determine IAA concentration from OD measurements of IAA-producing E. coli DH5α.
Figure 3: 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 4: 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 (Fig. 5).
Figure 5: 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 6: 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 (Fig. 6). 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.
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.
[1] Ball, E(1938) Heteroauxin and the growth of Escherichia coli. Journal of Bacteriology 36(5):. 559-565.