Difference between revisions of "Part:BBa K4239003"
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<h2>Description</h2> | <h2>Description</h2> | ||
| − | <p><i>fiatluxA</i> is | + | <p><i>fiatluxA</i> is to be used with |
| − | <i>fiatluxB</i> <a href="https://parts.igem.org/Part: | + | <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 | + | It codes for a subpart of the luciferase protein. With the subpart encoded by <i>fiatluxB</i>, they form the luciferase protein.</p> |
<p>Luciferase has as substrats FMNH2, O2 and Fatty aldehydes, and produces H20, Fatty Acids and FMN and emits luminescence.</p> | <p>Luciferase has as substrats FMNH2, O2 and Fatty aldehydes, and produces H20, Fatty Acids and FMN and emits luminescence.</p> | ||
| − | <p>The | + | <p>The system <i>fiatluxA/fiatluxB</i> is to be used with |
| − | <i>fiatluxC</i> <a href="https://parts.igem.org/Part: | + | <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: | + | <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: | + | 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> | operon.</p> | ||
| − | <p><i> | + | <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 | + | <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> |
<br> | <br> | ||
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<h2>Construction</h2> | <h2>Construction</h2> | ||
| − | <p>The <i>ilux</i> operon was available in a pGEX plasmid. <i>fiatluxA, fiatluxB</i> and <i> | + | <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: | + | <i>fiatluxABE</i> <a href="https://parts.igem.org/Part:BBa_K4239007" class="pr-0" target="_blank">(BBa_K4239007)</a>. |
<br> | <br> | ||
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 504
- 1000INCOMPATIBLE 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.