Regulatory

Part:BBa_K3138000:Design

Designed by: Kamila Liman, Jagoda Pierścińska   Group: iGEM19_Wroclaw   (2019-10-15)
Revision as of 22:27, 16 October 2019 by Lilithin (Talk | contribs) (→‎Design Notes)


Metallothionein-IV promoter Uniprot Q9HFC9-1


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 95
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 95
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 95
    Illegal BglII site found at 380
    Illegal BglII site found at 492
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 95
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 95
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Characterization

The promoter region was used to induce lycopene biosynthesis by Yarrowia lipolytica after exposure of the cells to heavy metals (Cd, Cu, Pb) in the growth medium.

Figure 1. Relative expression of Y. lipolytica genes after exposure of cells to heavy metals (Cu, Cd, Pb).

Verification of metallotionein respons to heavy metals using real-time PCR

The gene responding to heavy metals was identified using real-time PCR followed by its promoter region sequence being used for further analysis. The cells were grown in Erlenmeyer flasks containing 50 ml of YPD medium. To identify genes reposnding to heavy metals three different ions in different concentrations were used: Cd (10 mM) Cu (28 mM), Pb (50 mM). The control medium in this experiment was YPD medium without any heavy metals. The concentration of metal ions were taken from the available literature [1]. Actine gene was used as a reference gene for qRT-PCR experiment. Five different genes were analyzed: YALI0A02416g, YALI0E08387g, YALI0F11275g, YALI0C18481g, YALI0C22394g.

The RNA was extracted from the cells harvested at 72h of the culture.

Results

BBa_K3138000 part shoved the highest expression level after exposure to Cd, Cu, Pb ions. The expression was 20-35 times higher comparing to the control experiment.

Lycopen biosynthesis experiment

Figure 1. The biomass of Y. lipolytica transformant with lycopene biosynthesis pathway cloned under the control of pYALI0C18481. A) Control, B) Cu, C) Cd, D) Pb.

Source

This part was isolated from chromosome C on Yarrowia lipolityca A101 genome.

References

  • García S, Prado M, Dégano R and Domínguez A (2002) A copper-responsive transcription factor, CRF1, mediates copper and cadmium resistance in Yarrowia lipolytica. Journal of Biological Chemistry 277(40): 37359–37368.