Difference between revisions of "Part:BBa K2971001"
 
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<partinfo>BBa_K2971001 short</partinfo>
 
<partinfo>BBa_K2971001 short</partinfo>
  
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This part is the gene crtI (dr0861) from the extremophile Deinococcus radiodurans and encodes
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phytoene desaturase (CrtI). CrtI catalyzes the desaturation of phytoene (a colorless compound) into
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lycopene (a deep red compound)[1]. The desaturation produces a polyene chromophore. It is this
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chromophore that is utilized in the biogenic solar cell (see design page) to absorb energy from light.
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The UiOslo 2019 team used this gene in a composite part (BBa_K2971004) to produce the red pigment lycopene, in
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<i>Escherichia coli</i>, by inserting it into a vector along with the genes <i>crtE</i> and <i>crtI</i>. This composite part produced red cells when expressed in Escherichia coli confirming that the part functions as intended.
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The part was cloned into an expression vector under the arabionose inducible promotor <i>araC</i>. Successful cloning was confirmed with colony PCR of transformed colonies (figure 1). Colonies that carried the insert was sequenced to check for the potential presence of mutations or frame-shifts.
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<img style="width:40% !important;" src="https://2019.igem.org/wiki/images/9/91/T--UiOslo_Norway--basicColonyPCR.png">
 
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This part is the gene crtE (dr1395) from the extremophile Deinococcus radiodurans. The gene encodes geranylgeranyl diphosphate synthase (GGPPS). GGPPS catalyze the condensation of farnesyl pyrophosphate (FPP) and isopentenyl pyrophosphate (IPP) into geranylgeranyl diphosphate (GGPP). GGPP is a precursor for the carotenoid biosynthetic pathway. The sequence has been codon optimized for expression in Escherichia coli and iGEM registry compatibility.
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<strong>Figure 1: Colony-PCR of cells transformed with our basic parts (crtE, crtI and crtB)</strong></br>
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</p>  
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Primers were designed to amplify the part of the intended plasmid that contains the insert.</br>
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Numbers denote the colony checked with the primers. 14 and 23 are PCR products from empty vectors that function as controls. Colony 1-12 and 15 shows that we had several colonies of crtE with the insert. 16-20 shows colonies carrying the crtB insert. 21 show that we had one colony with the insert.  
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We expressed the protein in <i>E. coli</i> (DH5&#945;)(figure 2). We detected a faint protein band during our SDS analysis; it is likely the expression was not well-optimized.
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<img style="width:40% !important;" src="https://2019.igem.org/wiki/images/0/07/T--UiOslo_Norway--SDS.png">
 
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The UiOslo 2019 team used this gene in a composite part to produce the red pigment lycopene, in Escherichia coli, by expressing it in a vector along with the genes crtB and crtI.  
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<strong>Figure 2: SDS-page of colonies expressing our basic parts</strong></br>
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The expression of our basic parts were tested in E.coli (DH5&#945;). We could detect a faint band of CrtE and CrtI.
 
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'''References'''
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1. Schaub P, Yu Q, Gemmecker S, Poussin-Courmontagne P, Mailliot J, McEwen AG, et al. (2012) On the Structure and Function of the Phytoene Desaturase CRTI from Pantoea ananatis, a Membrane-Peripheral and FAD-Dependent Oxidase/Isomerase. PLoS ONE 7(6): e39550. https://doi.org/10.1371/journal.pone.0039550
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 00:09, 22 October 2019


crtI from Deinococcus radiodurans

This part is the gene crtI (dr0861) from the extremophile Deinococcus radiodurans and encodes phytoene desaturase (CrtI). CrtI catalyzes the desaturation of phytoene (a colorless compound) into lycopene (a deep red compound)[1]. The desaturation produces a polyene chromophore. It is this chromophore that is utilized in the biogenic solar cell (see design page) to absorb energy from light.

The UiOslo 2019 team used this gene in a composite part (BBa_K2971004) to produce the red pigment lycopene, in Escherichia coli, by inserting it into a vector along with the genes crtE and crtI. This composite part produced red cells when expressed in Escherichia coli confirming that the part functions as intended.

The part was cloned into an expression vector under the arabionose inducible promotor araC. Successful cloning was confirmed with colony PCR of transformed colonies (figure 1). Colonies that carried the insert was sequenced to check for the potential presence of mutations or frame-shifts.


Figure 1: Colony-PCR of cells transformed with our basic parts (crtE, crtI and crtB)
Primers were designed to amplify the part of the intended plasmid that contains the insert.
Numbers denote the colony checked with the primers. 14 and 23 are PCR products from empty vectors that function as controls. Colony 1-12 and 15 shows that we had several colonies of crtE with the insert. 16-20 shows colonies carrying the crtB insert. 21 show that we had one colony with the insert.


We expressed the protein in E. coli (DH5α)(figure 2). We detected a faint protein band during our SDS analysis; it is likely the expression was not well-optimized.


Figure 2: SDS-page of colonies expressing our basic parts
The expression of our basic parts were tested in E.coli (DH5α). We could detect a faint band of CrtE and CrtI.

References

1. Schaub P, Yu Q, Gemmecker S, Poussin-Courmontagne P, Mailliot J, McEwen AG, et al. (2012) On the Structure and Function of the Phytoene Desaturase CRTI from Pantoea ananatis, a Membrane-Peripheral and FAD-Dependent Oxidase/Isomerase. PLoS ONE 7(6): e39550. https://doi.org/10.1371/journal.pone.0039550


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 955
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]