Difference between revisions of "Part:BBa K4239007"
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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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<p> pBAD18-<i>fiatluxABE</i> has a kanamycin resistance gene, which is used to select clones with the plasmid.</p>
  
  
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title="Construction of <i>fiatluxABE</i>">
 
title="Construction of <i>fiatluxABE</i>">
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<figcaption><strong> Figure 1: </strong>Construction of <i>fiatluxABE</i> </figcaption>
  
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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...!!!!!!!!!!!!!!!!!
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<p><strong>1. Verify the plasmid construction</strong></p>
  
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caractérisation dans E.coli DH5alpha...!!!!!!!!!!!!!!
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<p>To check that the new plasmids contains the genes <i>fiatluxABE</i>, we carry out a miniprep of the plasmid pBAD18-<i>fiatluxABE</i>. This last one is digested by two enzymes, EcoR1 and Pst1 (iGEM restriction sites), and a migration on agarose gel was realised. The expected fragment are :
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<li> Non Digested plasmid : 8833 bp </li>
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<li> Double Digested plasmid : 3439 bp + 5394 bp
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<p>The strip at 3439 bp indicates that <i>fiatluxABE</i> is in the plasmid.</p>
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<figure align="center">
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alt="parts5"
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title="Migration of agarose gel of the plasmid pBAD18-<i>fiatluxABE</i>">
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<figcaption><strong> Figure 1: </strong>Migration of agarose gel of the plasmid pBAD18-<i>fiatluxABE</i> </figcaption>
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<p><strong>2. Effect of silent mutations on the luminescence</strong></p>
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<p>Since mutations have been introduced in the <i>ilux</i> operon, the effect on bioluminescence had to be tested before reassembling <i>fiatluxABE</i> and <i>fiatluxCD</i> in the same plasmid. In order to check the bioluminescence, the two plasmids pACYDuet-1-<i>fiatluxCD</i> and pBAD18-<i>fiatluxABE</i> were inserted into <i>E.coli</i> DH5α by electrotransformation (8). After transformation, the bioluminescence of the transformants was well observed with CHEMIDOC(link), indicating that the genes <i>fiatluxABE</i> and <i>fiatluxCD</i> are expressed and functional (Figure 2).
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title="Luminescent E.coli DH5α containing pACYDuet-1-<i>fiatluxCD</i> and pBAD18-<i>fiatluxABE</i> captured with a highly-sensitive camera, without needing to add arabinose to induce the promoter (basal expression). Left: captured luminescence. Right: simple photo of the Petri dish">
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<figcaption><strong> Figure 2: </strong>Luminescent E.coli DH5α containing pACYDuet-1-<i>fiatluxCD</i> and pBAD18-<i>fiatluxABE</i> captured with a highly-sensitive camera, without needing to add arabinose to induce the promoter (basal expression). Left: captured luminescence. Right: simple photo of the Petri dish </figcaption>
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Revision as of 15:22, 11 October 2022


Enhanced units fiatluxABE


Description

fiatluxA (BBa_K4239003) and fiatluxB (BBa_K4239004) 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_K4239006) , 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.

pBAD18-fiatluxABE has a kanamycin resistance gene, which is used to select clones with the plasmid.


parts1
Figure 1: Construction of fiatluxABE


Characterization

1. Verify the plasmid construction

To check that the new plasmids contains the genes fiatluxABE, we carry out a miniprep of the plasmid pBAD18-fiatluxABE. This last one is digested by two enzymes, EcoR1 and Pst1 (iGEM restriction sites), and a migration on agarose gel was realised. The expected fragment are :

  • Non Digested plasmid : 8833 bp
  • Double Digested plasmid : 3439 bp + 5394 bp

The strip at 3439 bp indicates that fiatluxABE is in the plasmid.

parts5
Figure 1: Migration of agarose gel of the plasmid pBAD18-fiatluxABE

2. Effect of silent mutations on the luminescence

Since mutations have been introduced in the ilux operon, the effect on bioluminescence had to be tested before reassembling fiatluxABE and fiatluxCD in the same plasmid. In order to check the bioluminescence, the two plasmids pACYDuet-1-fiatluxCD and pBAD18-fiatluxABE were inserted into E.coli DH5α by electrotransformation (8). After transformation, the bioluminescence of the transformants was well observed with CHEMIDOC(link), indicating that the genes fiatluxABE and fiatluxCD are expressed and functional (Figure 2).

parts6
Figure 2: Luminescent E.coli DH5α containing pACYDuet-1-fiatluxCD and pBAD18-fiatluxABE captured with a highly-sensitive camera, without needing to add arabinose to induce the promoter (basal expression). Left: captured luminescence. Right: simple photo of the Petri dish


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.