Difference between revisions of "Part:BBa K1985009"

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Arslan, Engin et al. "Overproduction Of Thebradyrhizobium Japonicum C-Type Cytochrome Subunits Of Thecbb3oxidase Inescherichia Coli". Biochemical and Biophysical Research Communications 251.3 (1998): 744-747. Web. 17 Oct. 2016.
 
Arslan, Engin et al. "Overproduction Of Thebradyrhizobium Japonicum C-Type Cytochrome Subunits Of Thecbb3oxidase Inescherichia Coli". Biochemical and Biophysical Research Communications 251.3 (1998): 744-747. Web. 17 Oct. 2016.
  
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K1985009 parameters</partinfo>
 
<partinfo>BBa_K1985009 parameters</partinfo>
 
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Revision as of 13:49, 17 October 2016

AraC pBAD mamOPXT


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
    Illegal BamHI site found at 2081
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 4856
    Illegal AgeI site found at 979
    Illegal AgeI site found at 1946
    Illegal AgeI site found at 2030
    Illegal AgeI site found at 2939
    Illegal AgeI site found at 3600
    Illegal AgeI site found at 4169
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2561
    Illegal BsaI.rc site found at 1232
    Illegal SapI site found at 961
    Illegal SapI.rc site found at 4736

This is a composite part of part BBa_K1321333 (Imperial iGem, 2014), BBa_K1985006, BBa_K1985000, BBa_K1985002, BBa_K1985001 a . It was used in pSB1A3.

Usage and Biology

Usage: The mamO gene was used to initiate the formation of magnetite by "nucleating" the crystal particles, allowing further development. It was used in combination with the proposed electron transport complex of mamO, P and X in vivo to form magnetite in vivo.

BBa_K1321333 is a regulatory part is made up of the Arabinose-Inducible promoter, pBAD, and its transcriptional inhibitor/activator, AraC. It was used to give increased control over the expression of part BBa_K1985007.

The part was used in pSB1A3 rather than pSB1C3 as it was cotransformed with pec86, a cytochrome maturation factor, which was in a chloramphenicol resistant plasmid. Pec86 is a pACYC184 derivative containing the E.coli genes, ccmABCDEFGH, of the aeg operon, expressed from the tet promoter of the plasmid. The genes are essential for maturation of cytochromes c, i.e. for the covalent attachment of the heme to the protein.

Biology: For more information on the biology of mamO,P and X see parts BBa_K1985006, BBa_K1985000,BBa_K1985002 and BBa_K1985001 and for more information on the AraC pBAD promoter please see BBa_K1321333.

Validation

The part was first validated with a diagnostic restriction digest using EcoRI and PstI and agarose gel electrophoresis. The expected band sizes from the digest were: 5478 base pairs for the plasmid backbone and 2114 for the insert. A 1kB plus DNA marker was used to verify the sizes of the bands and it was confirmed that the correct plasmid had been produced.

Figure 1. Agarose gel of the restriction digest of BBa_K1985009 in pSB1A3, with EcoRI and PstI.

References

Arslan, Engin et al. "Overproduction Of Thebradyrhizobium Japonicum C-Type Cytochrome Subunits Of Thecbb3oxidase Inescherichia Coli". Biochemical and Biophysical Research Communications 251.3 (1998): 744-747. Web. 17 Oct. 2016.

Functional Parameters