Difference between revisions of "Part:BBa K4343135"

(→‎1.IDH2 Gene)
(→‎2.Delta-12 desaturase)
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        For the Contribution, we completed the experimental characterization of the previous parts （BBa_K2935068、BBa_K925000、BBa_K346085 and BBa_K2117000） about efficient production or accumulation in Yarrowia lipolytica. These investigations included the effect of IDH2 knockdown on lipid accumulation, verification of delta 12 desaturase function from Synechocystis sp PCC 6803, introduction of the strong promoter PT7 and measurement of the expression intensity of the TEF promoter under different conditions, and these data were added to the corresponding BioBricks. All of these may be helpful to other teams and we hope it will make some contribution to the iGEM community.
 
        For the Contribution, we completed the experimental characterization of the previous parts （BBa_K2935068、BBa_K925000、BBa_K346085 and BBa_K2117000） about efficient production or accumulation in Yarrowia lipolytica. These investigations included the effect of IDH2 knockdown on lipid accumulation, verification of delta 12 desaturase function from Synechocystis sp PCC 6803, introduction of the strong promoter PT7 and measurement of the expression intensity of the TEF promoter under different conditions, and these data were added to the corresponding BioBricks. All of these may be helpful to other teams and we hope it will make some contribution to the iGEM community.
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===2.Delta-12 desaturase===
 
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Delta 12 Desaturase (Δ12 desaturase), a trans-membrane enzyme, can introduce a double bond at the Δ-12 site in the hydrocarbon chain of oleic acid (C18:1, Δ9). This converts the substrate to linoleic acid (C18:2 Δ9,12), which is further used as a substrate to produce EPA in the alternating action of desaturases and elongases[2]. Δ12 desaturase from Synechocystis sp PCC 6803 (SyElo9) was registered in 2012. Here, we verified that SyElo9 had functional activity in Y.lipolytica. First, we knocked out the endogenous Δ12 desaturase (encoded by the Fad2 gene) in Y. lipolytica polf to facilitate the examination of the extension activity for SyElo9. The gas chromatography results showed that overexpression of SyElo9 in strain polf ΔylFad2 can convert oleic acid to linoleic acid (Fig 2). Further, the introduction of SyElo9 into the Y. lipolytica polf genome and fermentation revealed a significant increase in the proportion of linoleic acid, up to 59.895% (Fig 2). These results provide references for future iGEM team to select suitable sources of Δ12 desaturase.<br>
 
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<center>https://static.igem.wiki/teams/4343/wiki/imp-1010-2-3.png</center>
 
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<center>Fig 2. The fatty acid distribution in various strains</center>
 
  
 
===3.Ptet-RBS-T7polymerase===
 
===3.Ptet-RBS-T7polymerase===

Revision as of 14:21, 10 October 2022


Ptet-RBS-T7polymerase

        For the Contribution, we completed the experimental characterization of the previous parts （BBa_K2935068、BBa_K925000、BBa_K346085 and BBa_K2117000） about efficient production or accumulation in Yarrowia lipolytica. These investigations included the effect of IDH2 knockdown on lipid accumulation, verification of delta 12 desaturase function from Synechocystis sp PCC 6803, introduction of the strong promoter PT7 and measurement of the expression intensity of the TEF promoter under different conditions, and these data were added to the corresponding BioBricks. All of these may be helpful to other teams and we hope it will make some contribution to the iGEM community.

3.Ptet-RBS-T7polymerase

        T7 RNA polymerase (RNAP) from λ prophage can specifically recognize the T7 promoter (PT7) and achieve high gene expression at a transcriptional rate 8-fold higher than that of Escherichia coli RNAP. This expression system has been successfully introduced into a variety of yeast hosts and has driven the CRISPR-Cas9 system to enable gene editing. However, the above systems are expressed using constitutive promoters and do not allow for precise regulation[3].         Based on this, we introduced the component Ptet-RBS-T7polymerase into Y. lipolytica and tested its regulatory function. To ensure that this element is functional, we added nuclear localization sequences (BBa_K4343135) to both terminals of the T7 RNAP and integrated a green fluorescent protein expression cassette regulated by the PT7 in the genome (polf-T7RNAP-T7GFP or polf-T7GFP). The strain polf-T7RNAP-GFP exhibited brighter fluorescence when the anhydrotetracycline (aTC) inducer was added, with a more than 5-fold increase in unit fluorescence intensity after induction (Fig 3A). At the same time, the system has no significant growth burden on the host (Fig 3B). This shows that the optimised BBA_K346085 can work in lipolytic yeast and provides a reference for future iGEM teams.

imp-1010-2-4.png
imp-1010-2-5.png
Fig. 3. A. The GFP expression in the polf-T7GFP and polf-T7RNAP-T7GFP. B. The growth of Y. lipolytica and polf-T7RNAP-GFP.

Reference

[1] Morse, N. J., Wagner, J. M., Reed, K. B., Gopal, M. R., Lauffer, L. H., & Alper, H. S. (2018). T7 polymerase expression of guide RNAs in vivo allows exportable CRISPR-Cas9 editing in multiple yeast hosts. ACS synthetic biology, 7(4), 1075-1084.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]