Difference between revisions of "Part:BBa K2971000"
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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 frameshift.  
 
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 frameshift.  
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<img style="width:40% !important;" src="https://2019.igem.org/wiki/images/4/4e/T--UiOslo_Norway--EBIcolonyPCR.jpg">
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<strong>Figure 1: Colony-PCR of cells transformed with our basic parts (crE, crtI and crtB)</strong></br>
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Primers were designed to amplify the part of the intented plasmid that contains the insert.</br>
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Numbers denote the colony checked with the primers.
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</html>
  
 
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.  
 
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.  

Revision as of 23:29, 21 October 2019


crtE from Deinococcus radiodurans

This part is the gene crtE (dr1395) from the extremophile Deinococcus radiodurans and encodes geranylgeranyl diphosphate synthase (GGPPS). GGPPS catalyze the condensation of farnesyl pyrophosphate (FPP) and isopentenyl pyrophosphate (IPP) into geranylgeranyl diphosphate (GGPP)[1]. GGPP is a precursor for the carotenoid biosynthetic pathway. The sequence has been codon optimized for expression in Escherichia coli and iGEM registry compatibility.

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 crtB 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 frameshift.

Figure 1: Colony-PCR of cells transformed with our basic parts (crE, crtI and crtB)
Primers were designed to amplify the part of the intented plasmid that contains the insert.
Numbers denote the colony checked with the primers.

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.


References

1. Liu, C., Sun, Z., Shen, S., Lin, L., Li, T., Tian, B., & Hua, Y. (2014). Identification and characterization of the geranylgeranyl diphosphate synthase in Deinococcus radiodurans. Letters in Applied Microbiology, 58(3), 219-224. doi:10.1111/lam.12181



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 841
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 43