Part:BBa_K5288009
Expression cassette for BADH protein
Usage and Biology
This part contains the linear sequence of BADH nucleotide optimized for E. coli. It incorporates some of the most efficient biobricks as described below. The T7 promoter with LacO regulations BBa_J435350, we selected this part to ensure a high translation of mRNA with a regulated expression. The high strength RBS BBa_J428038, we selected this part to guarantee an efficient binding to the ribosome and therefore an efficient traduction. Finally, the triple terminator BBa_J435371, we selected this part to warrant a precise termination of the translation of the mARN and consequently enhance protein expression. For our plasmid construction, we used the medium copy OriV9/Kan R backbone BBa_J428341, we selected this backbone to guarantee a more precise and stable genetic expression. This part contains part BBa_K5288003 which catalyzes the second step of glycine betaine biosynthesis, via choline [1], [2]. This part has a length of 2159 bp.
Betaine aldehyde dehydrogenase (BADH) consists of three domains; an N-terminal NAD+ binding domain, an oligomerization domain, and a C-terminal catalytic domain [3], [4], [5]. The enzyme has a length of 510 amino acids and a molecular weight of 55.61 kDa. The part is adapted to the Golden Gate cloning method.
We intend to use BADH as a key enzyme for the biosynthesis of the osmolyte and osmoprotectant; GB. As mentioned previously, BADH catalyzes the second step in the biosynthesis of GB. By this, we intend to ensure a better resistance to drought and caloric stress on corn. GB is an environmentally safe, non-toxic soluble compound [6]. GB stabilizes membranes and protects proteins while being under dehydration stress [7]. Furthermore, GB improves osmotic adjustment and stabilizes subcellular structures, preserving the thermodynamic stability of macromolecules and reversing protein misfolding without compromising native protein functionality. The high solubility and hydrophilicity of GB aid in maintaining the water contents of plants as much as possible in arid environments [8]. Even more, GB stimulates the production of ROS-scavenging enzymes. GB accumulation, in maize, is demonstrated to enhance the germination process and increase the number of antioxidant-related enzymes [9]. This will lead to a novel non-transgenic or synthetic solution to losses on corn production globally due to abiotic stress.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 323
Illegal EcoRI site found at 1833
Illegal XbaI site found at 96
Illegal PstI site found at 1760 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 323
Illegal EcoRI site found at 1833
Illegal NheI site found at 612
Illegal NheI site found at 861
Illegal PstI site found at 1760
Illegal NotI site found at 1975 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 323
Illegal EcoRI site found at 1833
Illegal BglII site found at 30
Illegal BamHI site found at 1827
Illegal XhoI site found at 1984 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 323
Illegal EcoRI site found at 1833
Illegal XbaI site found at 96
Illegal PstI site found at 1760 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 323
Illegal EcoRI site found at 1833
Illegal XbaI site found at 96
Illegal PstI site found at 1760 - 1000COMPATIBLE WITH RFC[1000]
Assembly
This part was assembled using type IIS Golden Gate assembly, as the iGEM community recommends. The basic parts (level 0), were flanked using type IIS enzyme; BsaI. The assembled level 1 construct has a length of 5117 bp.
Induction and expression
This part is still in progress to successfully express in E. coli BL21 (DE3)
References
[1] Z. Xu et al., “Glycinebetaine Biosynthesis in Response to Osmotic Stress Depends on Jasmonate Signaling in Watermelon Suspension Cells,” Front. Plant Sci., vol. 9, p. 1469, Oct. 2018, doi: 10.3389/fpls.2018.01469. <p> [2] S. S. Krishnamurthi, S. Kuttan, T. Nooruddin, and A. Parida, “The Role of the Overexpression of Suaeda maritima Choline Monooxygenase and Betaine Aldehyde Dehydrogenase cDNAs in the Enhancement of Salinity Tolerance in Different Strains of,” vol. 12, no. 1, 2019.
[3] M. Chen et al., “CRISPR/Cas9-Mediated Targeted Mutagenesis of Betaine Aldehyde Dehydrogenase 2 (BADH2) in Tobacco Affects 2-Acetyl-1-pyrroline,” Agronomy, vol. 14, no. 2, p. 321, Feb. 2024, doi: 10.3390/agronomy14020321.
[4] N. A. Stephens-Camacho, A. Muhlia-Almazan, A. Sanchez-Paz, and J. A. Rosas-Rodríguez, “Surviving environmental stress: the role of betaine aldehyde dehydrogenase in marine crustaceans,” Invertebr. Surviv. J., vol. 12, no. 1, Art. no. 1, Feb. 2015.
[5] C. Muñoz‐Bacasehua et al., “Heterogeneity of active sites in recombinant betaine aldehyde dehydrogenase is modulated by potassium,” J. Mol. Recognit., vol. 33, no. 10, p. e2869, Oct. 2020, doi: 10.1002/jmr.2869.
[6] S. Ali et al., “Glycine Betaine Accumulation, Significance and Interests for Heavy Metal Tolerance in Plants,” Plants, vol. 9, no. 7, p. 896, Jul. 2020, doi: 10.3390/plants9070896.
[7] P. Suraninpong, K. Thongkhao, A. M. Azzeme, and P. Suksa-Ard, “Monitoring Drought Tolerance in Oil Palm: Choline Monooxygenase as a Novel Molecular Marker,” Plants, vol. 12, no. 17, p. 3089, Aug. 2023, doi: 10.3390/plants12173089.
[8] M. Bai et al., “Transcriptome expression profiles reveal response mechanisms to drought and drought-stress mitigation mechanisms by exogenous glycine betaine in maize,” Biotechnol. Lett., vol. 44, no. 3, pp. 367–386, Mar. 2022, doi: 10.1007/s10529-022-03221-6.
[9] M. Niazian, S. A. Sadat-Noori, M. Tohidfar, S. M. M. Mortazavian, and P. Sabbatini, “Betaine Aldehyde Dehydrogenase (BADH) vs. Flavodoxin (Fld): Two Important Genes for Enhancing Plants Stress Tolerance and Productivity,” Front. Plant Sci., vol. 12, p. 650215, Apr. 2021, doi: 10.3389/fpls.2021.650215.
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