Contribution to auxin biosynthesis gene pathway

We have added additional information on the main page of the BBa_K3247007 part in the iGEM registry. Click on the link below to access this page on the iGEM registry:

In order to better characterize the indole-3-acetic acid (IAA) metabolic pathway that we are using in Escherichia coli, we have added documentation with new information learnt from the literature about an iGEM part that has been used previously by other teams, to induce IAA secretion. The information we have added is outlining the presence of the IAM pathway in bacteria and in every plant species. Moreover, we have outlined the impact of the genes iaaM and iaaH, that are essential enzymes in the IAM pathway towards producing IAA. We focused on the iaaM gene and the correlated IAM intermediate. Based on our literature review, the indole-3-acetamide (IAM) pathway that induces the biosynthesis of IAA from tryptophan is not specific only to bacteria, but IAM is also found in multiple plant species as the intermediates (IAM) and enzymes (indole-3-acetamide hydrolase) within the pathway are found in numerous species around the plant kingdom (such as Nicotiana tabacum, Citrus unshiu, rice etc.)(Sanchez-Parra et al., 2014). The IAM intermediate within the pathway has a major role in interfering with the conversion of the tryptophan to IAA that is realized by the IAA synthase complex in a vitro setting (Abu-Zaitoon et al., 2016). The IAA synthase complex facilitates the commitment of the pathway towards the IAA production through the formation of a tight metabolite channel (Pollmann et al., 2009). Therefore, the IAM pathway is important as IAA is implicated majorly in all aspects of plant growth and development as a regulator. The IAM pathway has been characterized best mainly in bacteria (Figure 1), the IAA biosynthesis process being influenced by different environmental factors such as acidic pH, osmotic stress carbon limitation or by genetic factors such as the location of the iaaM and iaaH genes in the genome. (Spaepen et al., 2007).

The existence of the pathway within the plant species has been confirmed through the transfection of a plant species with the plant pathogen Agrobacterium rhizogenes that is capable of tumorigenesis, the induced tumors being a result of the bacterial secreted IAA. The plant pathogen Agrobacterium rhizogenes, through containing a large-root inducing (Ri) plasmid, produces a hairy-root disease . In hairy roots, the IAA that enables the aforementioned growth is produced from the transformation of the Trp through the expression of the iaaM and iaaH genes that are present within a portion of the Ri plasmid that transferred to the infected host cell (Mano and Nemoto, 2012). To investigate the role of the IAA biosynthesis in the plant cell division that takes place in the meristematic regions of the plant is the Tobacco (Nicotiana tabacum) as its Bright Yellow-2 (BY-2) cells proliferate rapidly in the presence of only auxin the cell medium (Mano and Nemoto, 2012). In the case of the transgenic tobacco Bright Yellow-2 cell line formed with the induced Ri plasmid through the infection of the plant species with the pathogenic Agrobacterium rhizogenes, the overexpression of the iaaM gene alone is suffice to induce the growth of the transgenic tobacco line in the absence of IAA and in the presence of a lower concentration of IAM (10-5M) than the normal level. Subsequently, it has been deduced that the growth of the transgenic BY-2 cell line in the absence of auxin is because of the overexpression of the iaaM gene within the RI plasmid. Moreover, this permitted the indole-3-acetamide hydrolase gene to be isolated from Nicotiana sp. which was subsequently named NtAMI1 (Nemoto et al., 2009). As the transgenic cell line was placed subsequently in a IAM-containing medium, but where the NtAMI1 has been suppressed via RNA interference (RNAi), the cell line was completely inhibited, demonstrating the importance of the iaaM gene and the IAM intermediate compound.

Figure 1: The indole-3-acetamide pathway in bacteria (Duca et al., 2014)

Microalgae Growth Model:

Our goal for this model was to model the initial growth of Scenedesmus sp. LX1 and to assess what metabolite would be the limiting factor if we were to implement BloomAid. We utilised a modified monod type growth model equation to model the initial growth of Scenedesmus sp. LX1 under multiple limiting factors inside of wastewater medium. We modified it to add a function for growth of light intensity. We used two main limiting metabolites, Nitrogen and Phosphate as these were the main metabolites, limited in wastewater. This showed us that the specific growth rate for Scenedesmus was 0.4h-1 and when altering parameters we found Nitrogen was the main limiting factor to the initial growth of Scenedesmus therefore in our implementation we could recommend that nitrates be artificially added to the bioreactor to improve the initial growth rate.

Leakage Model:

Our Leakage model was designed to give us insight into the safety implementations of our project. It will inform us of the concentrations of IAA, N and P at different distances along the river and times. From this model we found that phosphate is the biggest threat during a leakage as it leaves the river much slower than the other compounds and is dumped at higher concentrations. However, the model can be performed with any river and contaminants. To find the MATLAB code files containing our model please see our modeling page here :