Difference between revisions of "Part:BBa K4239007"
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<h2>Construction</h2>  
 
<h2>Construction</h2>  
  
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<p>The <i>ilux</i> operon was available in a pGEX plasmid. <i>fiatluxA, fiatluxB</i> and <i>fiatluxE</i> were directly constructed together in <i>fiatluxABE</i>. Igem restriction sites were successfully removed in the <i>iluxABE</i> genes by following these steps: DNA extraction, PCR directed mutagenesis, agarose gel analysis with green gel, and gel purification. An overlap PCR was performed to reconstitute <i>iluxABE</i> fragments which had been cut by the restriction enzymes. The part is now called <i>fiatluxABE</i>.
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This part was then cloned and transformed plasmids in E.coli DH5α:
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<ul>
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  <li>: in a pSB1C3 (already in iGEM biobrick format), without promotor</li>
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  <li>: in a pBAD18, under the control of the arabinos inductible promotor pBAD.</li>
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</ul>
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</p>
  
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resultats de l'overlap PCR , j'ai pas trop compris ce qui a était fait...!!!!!!!!!!!!!!!!!
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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_K239007" class="pr-0" target="_blank">(BBa_K239007)</a>
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(ori colE1) which carries the resistance to kanamycin.</p>
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<br>
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<p align="center">
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<img
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alt="image_name"
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src="https://static.igem.wiki/teams/4239/wiki/parts/parts1.png"
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width="800"
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title="Construction of <i>fiatluxABE</i>">
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</p>
  
 
<br>
 
<br>
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<h2>Characterization</h2>  
 
<h2>Characterization</h2>  
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resultats de l'overlap PCR , j'ai pas trop compris ce qui a était fait...!!!!!!!!!!!!!!!!!
  
 
caractérisation dans E.coli DH5alpha...!!!!!!!!!!!!!!
 
caractérisation dans E.coli DH5alpha...!!!!!!!!!!!!!!

Revision as of 09:50, 10 October 2022


Enhanced units fiatluxABE


Description

fiatluxA (BBa_K239003) and fiatluxB (BBa_K239004) are made to be used together. They code for the luciferase protein.

Luciferase has as substrats FMNH2, O2 and Fatty aldehydes, and produces H20, Fatty Acids and FMN and emits luminescence.

The systeme fiatluxA/fiatluxB is made to be used with fiatluxC, fiatluxD and fiatluxE (BBa_K239006) , gathered in the fiatluxCDABE operon.

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.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2853
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 504
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1023


Construction

The ilux operon was available in a pGEX plasmid. fiatluxA, fiatluxB and fiatluxE were directly constructed together in fiatluxABE. Igem restriction sites were successfully removed in the iluxABE genes by following these steps: DNA extraction, PCR directed mutagenesis, agarose gel analysis with green gel, and gel purification. An overlap PCR was performed to reconstitute iluxABE fragments which had been cut by the restriction enzymes. The part is now called fiatluxABE. This part was then cloned and transformed plasmids in E.coli DH5α:

  • : in a pSB1C3 (already in iGEM biobrick format), without promotor
  • : in a pBAD18, under the control of the arabinos inductible promotor pBAD.

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_K239007) (ori colE1) which carries the resistance to kanamycin.


image_name



Characterization

resultats de l'overlap PCR , j'ai pas trop compris ce qui a était fait...!!!!!!!!!!!!!!!!! caractérisation dans E.coli DH5alpha...!!!!!!!!!!!!!!

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.