Difference between revisions of "Part:BBa K4239006"
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<p>pACYDuet-1-J23117-fiatluxCD was requested because its origin of replication (p15A) and its resistance to chloramphenicol makes it compatible in the same bacterium with the plasmid pBAD18-fiatluxABE <a href="https://parts.igem.org/Part:BBa_K4239007" class="pr-0" target="_blank">(BBa_K4239007)</a>
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(ori colE1) which carries the resistance to kanamycin. The goal is to insert both plasmids in the same bacteria, to gather the entire <i>fiatluxCDABE</i> operon</p>
  
 
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Revision as of 13:15, 11 October 2022


Enhanced luciferase substrate forming subunits fiatluxCD and the promoter J23117


Description

fiatluxC and fiatluxD are made to be used with fiatluxE. They code for a fatty acid reductase. With the subparts coding from fiatluxE, they form a complex that recycles fatty acids to fatty aldehydes. Fatty aldehydes will be used as a substrat for the luciferase protein.

The systeme fiatluxC/fiatluxD/fiatluxE is made to be used with fiatluxA and fiatluxB, gathered in the fiatluxCDABE operon. fiatluxA and fiatluxB code for the luciferase protein

Fiatlux genes come from ilux genes (C, D, A, B, E). They were modified to remove every Igem restriction site (EcoR1, Xba1, Spe1 and Pst1) included in genes. They were also adapted to include the biobrick format.

The ilux operon was born from a mutated natural luminescence operon present in the bacteria P.luminescens: the lux operon. These mutations were error-prone PCR induced according to Gregor et al.’s study in 2018 (Gregor et al. 2018). The aim was to create a system of genes that produced more light than the lux system.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 50
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1366


Construction

FiatluxCD was directly synthesized with the promoter J23117 (BBa_J23117). It is a constitutive and weak promoter, so no inducer needs to be added. FiatluxCD and their promoter were ordered in the plasmid pACYDuet-1, a p15A low-copy plasmid, and transformed it into E.coli DH5α.

parts2
Figure 1: Construction of fiatluxCD

pACYDuet-1-J23117-fiatluxCD was requested because its origin of replication (p15A) and its resistance to chloramphenicol makes it compatible in the same bacterium with the plasmid pBAD18-fiatluxABE (BBa_K4239007) (ori colE1) which carries the resistance to kanamycin. The goal is to insert both plasmids in the same bacteria, to gather the entire fiatluxCDABE operon


Characterization

GEL DE MIGRATION POUR VERIFIER QUE C4EST BIEN CD

References

Gregor C, Gwosch KC, Sahl SJ, Hell SW. Strongly enhanced bacterial bioluminescence with the ilux operon for single-cell imaging. Proc Natl Acad Sci U S A. 2018 Jan 30;115(5):962-967. doi: 10.1073/pnas.1715946115. Epub 2018 Jan 16. PMID: 29339494; PMCID: PMC5798359.