Difference between revisions of "Part:BBa K3247007"

Revision as of 17:55, 30 September 2022

Para-B0034-iaaMH-L3S1P00

This cassette contains a Pcon-araC promoter, an Elowitz RBS (BBa_B0034), the genes iaaM and iaaH which together produce IAA, and a terminator. IAA (indole-3-acetic acid) or more commonly known as auxin is a plant hormone that increases root surface area and length and stimulates plant growth. There are many biosynthesis pathways to produce IAA from tryptophan, but this circuit uses two enzymes: iaaM and iaaH. The enzyme iaaM (tryptophan-2-monooxygenase) converts tryptophan into IAM (indole-3-acetamide) and the enzyme iaaH (indoleacetamide hydrolase) converts IAM into IAA (indole-3-acetic acid).

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 507
Illegal BamHI site found at 26
Illegal BamHI site found at 1452
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 214
Illegal NgoMIV site found at 2803
1000
COMPATIBLE WITH RFC[1000]

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 IAA (indole-3-acetic acid) or more commonly known as auxin is a plant hormone that increases root surface area and length and stimulates plant growth. There are many biosynthesis pathways to produce IAA from tryptophan, but this circuit uses two enzymes: iaaM and iaaH.
 The enzyme iaaM (tryptophan-2-monooxygenase) converts tryptophan into IAM (indole-3-acetamide) and the enzyme iaaH (indoleacetamide hydrolase) converts IAM into IAA (indole-3-acetic acid).
 <!-- Add more about the biology of this part here
 ===Usage and Biology===
+The Team’s Manchester project involves the biosynthesis of auxin in order to induce growth of microalgae within a co-cultivation system that we want to establish. 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 every plant species, as well as the impact of the genes iaaM and iaaH, that are essential enzymes in the IAM pathway towards producing IAA, in plants, focusing on the iaaM gene and the correlated IAM intermediate.
+The 2019 Rice University Thermoplant team has used the BBa_K3247007 part from the iGEM registry in order to induce the biosynthesis of IAA within the E.coli through the expression of the iaaM and iaaH.The iaaM gene encodes the tryptophan-2-monooxygenase that converts Trp to IAM and the iaaH gene encodes the indole-3-acetamide hydrolase which converts IAM to IAA. They haven’t provided clear evidence on the amount of secreted auxin that would enable the plant root growth as they only used a control experiment with a fixed amount of IAA that would be used for further comparison with their enzyme circuit-inducing root growth. Moreover, the team hasn’t added information about the IAM pathway. Based on our literature findings, the IAM pathway is not specific only to bacteria, but the IAM intermediate is also found in multiple plant species in different concentrations, IAM interfering with the conversion of the tryptophan to IAA though the action of the IAA synthase complex (Zaitoon et.al., 2016) (https://doi.org/10.1590/1678-4324-2016150677). Therefore, the importance of the IAM pathway is around the presence within numerous species around the plant kingdom (such as Nicotiana tabacum, Citrus unshiu, rice etc )of IAM as an intermediate within the IAA synthesis and the indole-3-acetamide hydrolase as an essential enzyme in the conversion of IAM to IAA.
+As previously mentioned, it has been thought that the IAA pathway is strictly related to bacteria, as there wasn’t any information regarding  the presence of the pathway in plants. 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 it’s transferred to the host cell that it’s infected (Mano and Nemoto, 2012) (https://doi.org/10.1093/jxb/ers091).
+A relevant example in order to investigate the role of auxin 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. 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) (https://febs.onlinelibrary.wiley.com/doi/full/10.1016/j.febslet.2008.12.049) . 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.
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 ===<span class='h3bb'>Sequence and Features</span>===