Difference between revisions of "Part:BBa K515100"

Revision as of 00:44, 6 October 2011

IAA biosynthetic genes under control of the Pveg2 promoter

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 547
Illegal BamHI site found at 1492
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 254
Illegal NgoMIV site found at 2835
1000
COMPATIBLE 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].

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 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.

1.2 HPLC

Since the BL21 strain of E. coli seemed to be saturating the Salkowski reagent we had to look for alternative quantification methods to measure the amount of IAA that was being produced by the BL21 cells. We therefore grew up a control culture as well as our Auxin Xpress culture in LB broth without supplementing it with tryptophan and then extracted the IAA. We obtained a small peak in the HPLC Auxin Xpress cell line and no peak in the control. However, the peak appeared about five minutes later than our standard peaks. This could be because we had to replace the buffer after running the first sets of experiments and the buffer might have had a slightly different composition or the sample that was extracted was not dried completely and residue ethyl acetate contaminated the sample. While this preliminary result seems promising, there was not enough time for data analysis and further experimentation.

Figure 8: Image comparing the results of Salkowski before extraction and after extraction. The tube furthest to the left was the cell filtrate of the control, the one to the right of it was the cell filtrate of our Auxin Xpress cells. The eppendorf furthest to the right was the extract of the filtrate of our Auxin Xpress cells and the one to the left of it was the control. (Picture by Imperial College London iGEM team 2011).

We also tested the extracts with Salkowski reagent and saw a striking colour difference between the two samples (see Figure 8). We will have to repeat the HPLC experiments to obtain more data. In the future, we would like to run the BL21 cell lines with tryptophan supplemented media to see if the size of the peak increases and compare it with DH5α cell lines.

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.

@@ Line 22: / Line 22: @@
 <p> 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) <a href="https://parts.igem.org/Part:BBa_K515000">BBa_K515000</a>, 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) <a href="https://parts.igem.org/Part:BBa_K515001">BBa_K515001</a>. There are several different pathways that produce indole-3-acetic acid [1]. IaaM and IaaH originate from <i>P. savastanoi</i> and have been expressed in <i>E. coli</i> previously, and shown to secrete auxin into the cell supernatant [2].</p>
-[[File:M_Mechanism.png‎|680px]]
-[[File:M_P1_S1.png|680px]]
-[[File:M_P2_S2.png‎|680px]]
-''Fig. 2 Sequence alignment of wild type ptsG/SgrS pair and its mutant complementary pairs.''
-''(A) The partial complementary region of ptsG (wt) mRNA and its corresponding sRNA SgrS.''
-''(B) The complementary pair of ptsG1 mRNA and corresponding SgrS1.''
-''(C) Another complementary pair site-mutant version of ptsG2 mRNA and corresponding SgrS2.''
-pathway which involves genes IaaM and IaaH to convert tryptophan to IAA via the IAM intermediate. </i></p>
 </div>
 <h2>Experimental Data</h2>