Difference between revisions of "Part:BBa K4273020"
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| − | + | ==Usage and Biology== | |
We selected promoters pTDH3, pPGK1, and pTEF2 due to their stability expression in S. cerevisiae (Apel et. al., 2016). These promoters are shown to have stable and strong expression in YPD culture mediums. Among the three, pTDH3 has highest stability and strength, followed by pPGK1. For expression of AGL and AlaL enzymes, we used pTDH3 for AGL and pPGK1 for AlaL. In order to optimize our production, we inserted this part into the yeast’s genome at position 106, chromosome I (Apel et. al., 2016). | We selected promoters pTDH3, pPGK1, and pTEF2 due to their stability expression in S. cerevisiae (Apel et. al., 2016). These promoters are shown to have stable and strong expression in YPD culture mediums. Among the three, pTDH3 has highest stability and strength, followed by pPGK1. For expression of AGL and AlaL enzymes, we used pTDH3 for AGL and pPGK1 for AlaL. In order to optimize our production, we inserted this part into the yeast’s genome at position 106, chromosome I (Apel et. al., 2016). | ||
| − | + | ==Experiment== | |
Design and Absorbance of Nine AGL-AlaL Combinations | Design and Absorbance of Nine AGL-AlaL Combinations | ||
Revision as of 10:55, 12 October 2022
pTDH3-Np5598-tTDH1-pPGK1-NlmysD-tPGK1
Np5598 is a gene found in cyanobacteria Nostoc punctiforme that encodes for AGL whereas NlmysD encodes for AlaL in Nostoc linckia. AGL-AlaL allows 4-DG to be converted into the MAAs shinorine or porphyra-334. In our experiment, we found that AlaL encoded by NlmysD has a strong preference toward the amino acid threonine. Therefore, this part could efficiently produce the MAA porphyra-334 primarily, making it the first successful case of producing pure samples of porphyra-334. This part is within our part collection that allows efficiently production of MAAs in S. cerevisiae. Our part collection contains necessary genes to produce gadusol and the MAAs shinorine, porphyra-334, and palythine at a high rate. Xyl1, Xyl2, and Xyl3 are genes that allow S. cerevisiae to utilize xylose to produce S7P. DDGS and OMT converts S7P to the precursor of MAA, 4-deoxygadusol (4-DG). AGL converts 4-DG into M-glycine (MG). AlaL, by adding either serine or threonine, produces shinorine and porphyra-334, respectively. MysH could be added to the circuit of shinorine to produce palythine. S7P could also be converted into gadusol under the catalyzation of EEVS and M-Tox. In this part collection, we included multiple pathways and methods to increase the production of the upstream S7P and downstream MAAs. This part collection can provide inspiration and efficient methods to utilize the penta phosphate pathway or to produce other types of MAAs in S. cerevisiae for other teams.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3423
Illegal XhoI site found at 2075 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2321
Illegal BsaI.rc site found at 3433