Difference between revisions of "Part:BBa K3247007"
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===Usage and Biology===
 
===Usage and Biology===
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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.  
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Group: Manchester iGEM Team 2022
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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.
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Authors: Aretia-Teodora Malacopol, Franco Herrera
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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).
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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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Summary: We have added information about the iaaH and iaaM genes within the indole-3-acetamide (IAM) biosynthesis pathway followed by the biosynthesis of indole-3-acetic acid (IAA) in plants. Moreover, we have better characterized the functionality of the genes and their source organisms. In order to better characterize the indole-3-acetic acid (IAA) metabolic pathway which can be incorporated in Escherichia coli, we have added documentation learnt from the literature, to allow IAA production. The added information demonstrates the presence of the IAM pathway in bacteria and even in some plant species (Pollmann et.al., 2009). Moreover, we have outlined the impact of the genes iaaM and iaaH, which produce essential enzymes in the IAM pathway.
 +
 
 +
We focused on the iaaM gene and the correlated IAM intermediate. The indole-3-acetamide (IAM) pathway which converts tryptophan to IAM is not unique to bacteria, but also found in multiple plant species, 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, or rice (Sanchez-Parra et.al., 2014).
 +
 
 +
The IAM intermediate within the pathway has a major role in the conversion of the tryptophan to IAA. This was demonstrated by the IAA synthase complex in a in vitro enzyme array (Abu-Zaitoon et.al., 2016), which shows that the IAA synthase complex facilitates IAA production through forming a tight metabolite channel (Pollmann et.al., 2009). The channel allows IAA secretion, thus allowing IAA to facilitate various aspects of plant growth and development. Therefore, the IAM pathway is important in producing IAM intermediate which forms the tight metabolite channel for IAA secretion.
 +
 
 +
The IAM pathway has been best characterized in bacteria, the IAA biosynthesis process can be influenced by different environmental factors, such as acidic pH, osmotic stress, carbon limitation, or genetic factors. For example, IAA production can be affected by the location of the iaaM and iaaH genes in the genome (Spaepen et.al., 2007). The existence of the pathway has been confirmed through the transfection of Nicotiana tabacum with the plant pathogen Agrobacterium rhizogenes that is capable of tumorigenesis, the induced tumors being a result of the bacterial secreted IAA (Mano and Nemoto, 2012). The Agrobacterium rhizogenes, containing a large-root inducing (Ri) plasmid, produces a hairy-root disease (Mano and Nemoto, 2012). 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). IAA biosynthesis takes place in the Nicotiana tabacum meristematic regions (Mano and Nemoto, 2012).  Nemoto et al (2009) suggest that Bright Yellow-2 (BY-2) cells proliferate rapidly in the presence of only auxin the cell medium. On the other hand,  transgenic Nicotiana tabacum Bright Yellow-2 cell line formed with the induced Ri plasmid through the infection of Agrobacterium rhizogenes, the overexpression of the iaaM gene alone is sufficient to induce the growth of the transgenic tobacco line in the absence of IAA and in the presence of a low concentration of IAM (10-5M). Subsequently, 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, which permitted the indole-3-acetamide hydrolase gene named NtAMI1. 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 (Nemoto et al, 2009).
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Reference list:
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Abu-Zaitoon, Y., Aladaileh, S., Al Tawaha, A.R. (2016). Contribution of the IAM Pathway to IAA Pool in Developing Rice Grains. Braz. arch. biol. technol. 59 https://doi.org/10.1590/1678-4324-2016150677
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Duca, D., Lorv, J., Patten, C. L., Rose, D., & Glick, B. R. (2014). Indole-3-acetic acid in plant-microbe interactions. Antonie van Leeuwenhoek, 106(1), 85–125. https://doi.org/10.1007/s10482-013-0095-y
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Mano, Y., Nemoto, K. (2012) The pathway of auxin biosynthesis in plants, Journal of Experimental Botany, Volume 63, Issue 8, Pages 2853–2872, https://doi.org/10.1093/jxb/ers091
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Nemoto, K., Hara, M., Suzuki, M., Seki, H., Muranaka, T. and Mano, Y.(2009), The NtAMI1 gene functions in cell division of tobacco BY-2 cells in the presence of indole-3-acetamide, FEBS Letters, 583, doi: 10.1016/j.febslet.2008.12.049
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Pollmann, S., Düchting, P., Weiler, E. W. (2009) Tryptophan-dependent indole-3-acetic acid biosynthesis by ‘IAA-synthase’ proceeds via indole-3-acetamide, Phytochemistry, Volume 70, Issue 4, https://doi.org/10.1016/j.phytochem.2009.01.021
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Sánchez-Parra B, Frerigmann H, Alonso MM, et al. Characterization of Four Bifunctional Plant IAM/PAM-Amidohydrolases Capable of Contributing to Auxin Biosynthesis. Plants (Basel). 2014;3(3):324-347. Published 2014 Aug 7. doi:10.3390/plants3030324
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Spaepen, S., Vanderleyden, J., Remans, R., Indole-3-acetic acid in microbial and microorganism-plant signaling, FEMS Microbiology Reviews, Volume 31, Issue 4, July 2007, Pages 425–448, https://doi.org/10.1111/j.1574-6976.2007.00072.x
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===<span class='h3bb'>Sequence and Features</span>===
 
===<span class='h3bb'>Sequence and Features</span>===
 
<partinfo>BBa_K3247007 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3247007 SequenceAndFeatures</partinfo>

Latest revision as of 22:15, 13 October 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).


Usage and Biology

Group: Manchester iGEM Team 2022 Authors: Aretia-Teodora Malacopol, Franco Herrera

Summary: We have added information about the iaaH and iaaM genes within the indole-3-acetamide (IAM) biosynthesis pathway followed by the biosynthesis of indole-3-acetic acid (IAA) in plants. Moreover, we have better characterized the functionality of the genes and their source organisms. In order to better characterize the indole-3-acetic acid (IAA) metabolic pathway which can be incorporated in Escherichia coli, we have added documentation learnt from the literature, to allow IAA production. The added information demonstrates the presence of the IAM pathway in bacteria and even in some plant species (Pollmann et.al., 2009). Moreover, we have outlined the impact of the genes iaaM and iaaH, which produce essential enzymes in the IAM pathway.

We focused on the iaaM gene and the correlated IAM intermediate. The indole-3-acetamide (IAM) pathway which converts tryptophan to IAM is not unique to bacteria, but also found in multiple plant species, 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, or rice (Sanchez-Parra et.al., 2014).

The IAM intermediate within the pathway has a major role in the conversion of the tryptophan to IAA. This was demonstrated by the IAA synthase complex in a in vitro enzyme array (Abu-Zaitoon et.al., 2016), which shows that the IAA synthase complex facilitates IAA production through forming a tight metabolite channel (Pollmann et.al., 2009). The channel allows IAA secretion, thus allowing IAA to facilitate various aspects of plant growth and development. Therefore, the IAM pathway is important in producing IAM intermediate which forms the tight metabolite channel for IAA secretion.

The IAM pathway has been best characterized in bacteria, the IAA biosynthesis process can be influenced by different environmental factors, such as acidic pH, osmotic stress, carbon limitation, or genetic factors. For example, IAA production can be affected by the location of the iaaM and iaaH genes in the genome (Spaepen et.al., 2007). The existence of the pathway has been confirmed through the transfection of Nicotiana tabacum with the plant pathogen Agrobacterium rhizogenes that is capable of tumorigenesis, the induced tumors being a result of the bacterial secreted IAA (Mano and Nemoto, 2012). The Agrobacterium rhizogenes, containing a large-root inducing (Ri) plasmid, produces a hairy-root disease (Mano and Nemoto, 2012). 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). IAA biosynthesis takes place in the Nicotiana tabacum meristematic regions (Mano and Nemoto, 2012). Nemoto et al (2009) suggest that Bright Yellow-2 (BY-2) cells proliferate rapidly in the presence of only auxin the cell medium. On the other hand, transgenic Nicotiana tabacum Bright Yellow-2 cell line formed with the induced Ri plasmid through the infection of Agrobacterium rhizogenes, the overexpression of the iaaM gene alone is sufficient to induce the growth of the transgenic tobacco line in the absence of IAA and in the presence of a low concentration of IAM (10-5M). Subsequently, 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, which permitted the indole-3-acetamide hydrolase gene named NtAMI1. 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 (Nemoto et al, 2009).

Reference list:

Abu-Zaitoon, Y., Aladaileh, S., Al Tawaha, A.R. (2016). Contribution of the IAM Pathway to IAA Pool in Developing Rice Grains. Braz. arch. biol. technol. 59 https://doi.org/10.1590/1678-4324-2016150677


Duca, D., Lorv, J., Patten, C. L., Rose, D., & Glick, B. R. (2014). Indole-3-acetic acid in plant-microbe interactions. Antonie van Leeuwenhoek, 106(1), 85–125. https://doi.org/10.1007/s10482-013-0095-y

Mano, Y., Nemoto, K. (2012) The pathway of auxin biosynthesis in plants, Journal of Experimental Botany, Volume 63, Issue 8, Pages 2853–2872, https://doi.org/10.1093/jxb/ers091

Nemoto, K., Hara, M., Suzuki, M., Seki, H., Muranaka, T. and Mano, Y.(2009), The NtAMI1 gene functions in cell division of tobacco BY-2 cells in the presence of indole-3-acetamide, FEBS Letters, 583, doi: 10.1016/j.febslet.2008.12.049

Pollmann, S., Düchting, P., Weiler, E. W. (2009) Tryptophan-dependent indole-3-acetic acid biosynthesis by ‘IAA-synthase’ proceeds via indole-3-acetamide, Phytochemistry, Volume 70, Issue 4, https://doi.org/10.1016/j.phytochem.2009.01.021


Sánchez-Parra B, Frerigmann H, Alonso MM, et al. Characterization of Four Bifunctional Plant IAM/PAM-Amidohydrolases Capable of Contributing to Auxin Biosynthesis. Plants (Basel). 2014;3(3):324-347. Published 2014 Aug 7. doi:10.3390/plants3030324 Spaepen, S., Vanderleyden, J., Remans, R., Indole-3-acetic acid in microbial and microorganism-plant signaling, FEMS Microbiology Reviews, Volume 31, Issue 4, July 2007, Pages 425–448, https://doi.org/10.1111/j.1574-6976.2007.00072.x

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]