Difference between revisions of "Part:BBa K4239007"
| Line 10: | Line 10: | ||
| − | <p><i>fiatluxA</i> <a href="https://parts.igem.org/Part: | + | <p><i>fiatluxA</i> <a href="https://parts.igem.org/Part:BBa_K4239003" class="pr-0" target="_blank">(BBa_K4239003)</a> |
| − | and <i>fiatluxB</i> <a href="https://parts.igem.org/Part: | + | and <i>fiatluxB</i> <a href="https://parts.igem.org/Part:BBa_K4239004" class="pr-0" target="_blank">(BBa_K4239004)</a> |
are made to be used together. They code for the luciferase protein. | are made to be used together. They code for the luciferase protein. | ||
<p>Luciferase has as substrats FMNH<sub>2</sub>, O<sub>2</sub> and Fatty aldehydes, and produces H<sub>2</sub>0, Fatty Acids and FMN and emits luminescence.</p> | <p>Luciferase has as substrats FMNH<sub>2</sub>, O<sub>2</sub> and Fatty aldehydes, and produces H<sub>2</sub>0, Fatty Acids and FMN and emits luminescence.</p> | ||
| − | <p>The systeme <i>fiatluxA/fiatluxB</i> is made to be used with <i>fiatluxC, fiatluxD and fiatluxE</i> <a href="https://parts.igem.org/Part: | + | <p>The systeme <i>fiatluxA/fiatluxB</i> is made to be used with <i>fiatluxC, fiatluxD and fiatluxE</i> <a href="https://parts.igem.org/Part:BBa_K4239006" class="pr-0" target="_blank">(BBa_K4239006)</a> |
, gathered in the <i>fiatluxCDABE</i> operon.</p> | , gathered in the <i>fiatluxCDABE</i> operon.</p> | ||
Revision as of 14:33, 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2853
- 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. 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.
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.