Difference between revisions of "Part:BBa K4239003"
 
(9 intermediate revisions by the same user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K4239003 short</partinfo>
 
<partinfo>BBa_K4239003 short</partinfo>
  
−
fiatluxA is made to be used with fiatluxB. It codes for a subpart of the luciferase protein. With the subpart coding from fiatluxD, they form the luciferase protein.
+
<html>
  
−
Luciferase has as substrats FMNH2, O2 and Fatty aldehydes, and produces H20, Fatty Acids and FMN and emits luminescence.
+
<br>
  
−
The systeme fiatluxA/fiatluxB is made to be used with fiatluxC, fiatluxD and fiatluxE, gathered in the fiatluxCDABE operon.
+
<h2>Description</h2>
  
−
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.
+
<p><i>fiatluxA</i> is to be used with
 +
<i>fiatluxB</i> <a href="https://parts.igem.org/Part:BBa_K4239004" class="pr-0" target="_blank">(BBa_K4239004)</a>.  
 +
It codes for a subpart of the luciferase protein. With the subpart encoded by <i>fiatluxB</i>, they form the luciferase protein.</p>
  
−
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.
+
<p>Luciferase has as substrats FMNH2, O2 and Fatty aldehydes, and produces H20, Fatty Acids and FMN and emits luminescence.</p>
  
 +
<p>The system <i>fiatluxA/fiatluxB</i> is to be used with
 +
<i>fiatluxC</i> <a href="https://parts.igem.org/Part:BBa_K4239001" class="pr-0" target="_blank">(BBa_K4239001)</a>,
 +
<i>fiatluxD</i> <a href="https://parts.igem.org/Part:BBa_K4239002" class="pr-0" target="_blank">(BBa_K4239002)</a>
 +
and <i>fiatluxE</i> <a href="https://parts.igem.org/Part:BBa_K4239005" class="pr-0" target="_blank">(BBa_K4239005)</a>, gathered in the <i>fiatluxCDABE</i> <a href="https://parts.igem.org/Part:BBa_K4239006" class="pr-0" target="_blank">(BBa_K4239006)</a>
 +
operon.</p>
  
−
===Usage and Biology===
+
<p><i>fiatlux</i> genes come from <i>ilux</i> 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.</p>
  
−
<!-- -->
+
<p>The <i>ilux</i> operon was born from a mutated natural luminescence operon present in the bacteria <i>P.luminescens</i>: the <i>lux</i> 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.</p>
−
<span class='h3bb'>Sequence and Features</span>
+
 
 +
<br>
 +
 
 +
</html>
 +
 
 +
<h2>Sequence and Features</h2>
 
<partinfo>BBa_K4239003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4239003 SequenceAndFeatures</partinfo>
 +
 +
<html>
 +
 +
<br>
 +
 +
<h2>Construction</h2>
 +
 +
<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. A classical 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>. This part was then cloned and transformed in a pSB1C3 (already in iGEM biobrick format) and pBAD18 (high-copy vector with an arabinose inducible promoter) plasmid in <i>E.coli</i> DH5α. More details about the construction are on the following page
 +
<i>fiatluxABE</i> <a href="https://parts.igem.org/Part:BBa_K4239007" class="pr-0" target="_blank">(BBa_K4239007)</a>.
 +
 +
<br>
 +
<br>
 +
 +
<h2>References</h2>
 +
 +
<p><i> 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.</i> </p>
 +
 +
<!===Usage and Biology===
 +
 +
<!-- -->
 +
 +
  
  

Latest revision as of 15:09, 12 October 2022


Enhanced luciferase substrate forming units fiatluxA


Description

fiatluxA is to be used with fiatluxB (BBa_K4239004). It codes for a subpart of the luciferase protein. With the subpart encoded by fiatluxB, they form the luciferase protein.

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

The system fiatluxA/fiatluxB is to be used with fiatluxC (BBa_K4239001), fiatluxD (BBa_K4239002) and fiatluxE (BBa_K4239005), gathered in the fiatluxCDABE (BBa_K4239006) 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
    COMPATIBLE WITH RFC[21]
  • 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. A classical 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 in a pSB1C3 (already in iGEM biobrick format) and pBAD18 (high-copy vector with an arabinose inducible promoter) plasmid in E.coli DH5α. More details about the construction are on the following page fiatluxABE (BBa_K4239007).

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.