Difference between revisions of "Part:BBa K3138000:Design"

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===Design Notes===
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<h2>Characterization</h2>
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'''Characterization'''
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<p>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. </p>
 
<p>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. </p>
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[[Image:Yali_qPCR.PNG|500px|thumb|Figure 1. ''Relative expression of Y. lipolytica genes after exposure of cells to heavy metals (Cu, Cd, Pb). '']]
 
[[Image:Yali_qPCR.PNG|500px|thumb|Figure 1. ''Relative expression of Y. lipolytica genes after exposure of cells to heavy metals (Cu, Cd, Pb). '']]
  
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''Verification of metallotionein respons to heavy metals using real-time PCR''
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<h3>Verification of metallotionein respons to heavy metals using real-time PCR</h3>
 
<p>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.</p>
 
<p>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.</p>
  
 
<p>The RNA was extracted from the cells harvested at 72h of the culture.</p>
 
<p>The RNA was extracted from the cells harvested at 72h of the culture.</p>
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<h3>Results</h3>
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<p>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.</p>
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<h2>Lycopen biosynthesis experiment</h2>
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[[Image:Yali_biomass.PNG|400px|thumb|Figure 2. ''The biomass of Y. lipolytica transformant with lycopene biosynthesis pathway cloned under the control of pYALI0C18481. A) Control, B) Cu, C) Cd, D) Pb. '']]
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<p>The JMP62 series plasmid for Y. lipolytica was used as a basic vector for lycopene biosynthesis. Four genes from the lycopene biosynthesis pathway: phytoene synthase, phytoene dehydrogenase, isopentenyl-diphosphate isomerase, geranylgeranyl diphosphate synthase were assembled as one expression cassette, with the expression of each of the genes being controlled by the promoter of YALI0C18481 gene. The expression cassette was inserted into the genome of Y. lipolytica A-101 strain.  The transformants of Y. lipolytica were cultured in YPG medium with and without (control) metal ions (Cd, Cu,  Pb). The results are shown on Fig. 2. </p>
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<h3>Results</h3>
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<p>The Y. lipolytica transformants with lycopene biosynthesis pathway controlled by pYALI0C18481 promoter proved the concept, that biosynthesis of this carotenoid may be induced by the presence of heavy metals in the growth medium (Fig. 2 B,C,D). In the control experiment, cells did not show any color after the culture (Fig. 2A).</p>
  
 
===Source===
 
===Source===
  
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This part was isolated from chromosome C on Yarrowia lipolityca A101 genome.
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This part was isolated from chromosome C on Yarrowia lipolityca E150 genome.
  
 
===References===
 
===References===
  
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* García S, Prado M, Dégano R and Domínguez A (2002) <i>A copper-responsive transcription factor, CRF1, mediates copper and cadmium resistance in Yarrowia lipolytica.</i> Journal of Biological Chemistry 277(40): 37359–37368.
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[1] García S, Prado M, Dégano R and Domínguez A (2002) <i>A copper-responsive transcription factor, CRF1, mediates copper and cadmium resistance in Yarrowia lipolytica.</i> Journal of Biological Chemistry 277(40): 37359–37368.

Latest revision as of 18:15, 20 October 2019


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]


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

The JMP62 series plasmid for Y. lipolytica was used as a basic vector for lycopene biosynthesis. Four genes from the lycopene biosynthesis pathway: phytoene synthase, phytoene dehydrogenase, isopentenyl-diphosphate isomerase, geranylgeranyl diphosphate synthase were assembled as one expression cassette, with the expression of each of the genes being controlled by the promoter of YALI0C18481 gene. The expression cassette was inserted into the genome of Y. lipolytica A-101 strain. The transformants of Y. lipolytica were cultured in YPG medium with and without (control) metal ions (Cd, Cu, Pb). The results are shown on Fig. 2.

Results

The Y. lipolytica transformants with lycopene biosynthesis pathway controlled by pYALI0C18481 promoter proved the concept, that biosynthesis of this carotenoid may be induced by the presence of heavy metals in the growth medium (Fig. 2 B,C,D). In the control experiment, cells did not show any color after the culture (Fig. 2A).

Source

This part was isolated from chromosome C on Yarrowia lipolityca E150 genome.

References

[1] 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.