Difference between revisions of "Part:BBa K2060001:Experience"
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===Applications of BBa_K2060001=== | ===Applications of BBa_K2060001=== | ||
| + | We developed the pBAD::mKeima construct as part of the Cardiff_Wales [http://2016.igem.org/Team:Cardiff_Wales/mkeima 'Fuel Project']. We aimed to induce expression from two strain of E.coli that either contained the previously characterised [https://parts.igem.org/Part:BBa_K325909 pBAD:LUXoperon] construct or our own mKeima construct. [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207116/ Fluorescence by Unbound Excitation from Luminescence (FUEL) reaction] describes a phenomonon whereby a light output can excite a unrelated fluorophore to produce light of a different wavelength. Therefore we aimed to induce a red-light output from cells expressing the mKeima protein when mixed with cells expressing the LUXoperon, which gives out blue light at approximately 480nm. | ||
| + | <p>As described on our [http://2016.igem.org/Team:Cardiff_Wales/mkeima Wiki] we were able to induce blue light production from LUXoperon using a range of arabinose concentrations. However our efforts to demonstrate FUEL were thwarted by our inability to induce an output from cells containing the mKeima construct, which when excited at 440nm emits [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2851791/ light at 620nm].</p> | ||
| + | <p>We attempted to induce pBAD:mKeima by growing bacteria at a range of temperatures and across a wide range of arabinose concentrations. Although our experimental control cells (which contained a pBAD:sfGFP construct) showed expression across these different parameters unfortunately we were unable to induce mKeima expression that resulted in a light output at 620nm.</p> | ||
| + | <hr> | ||
| + | <br> | ||
| + | <p>Our attempts to induce expression of mKeima included experiments in which bacteria were grown under the following conditions:</p> | ||
| + | <p>1. Grow o/n without induction. Subculture (1/10) for 2hr. Induce with arabinose at concentrations between 100uM-10mM.</p> | ||
| + | <p>2. Grow o/n at 20C, 25C or 37C with arabinose induction at concentrations between 5uM-50mM.</p> | ||
| + | <p>3. Grow o/n without induction. Immediately induce with arabinose at concentrations between 100uM-5mM. Incubale cultures for 2-5h at 20C or 37C.</p> | ||
| + | |||
| + | <hr> | ||
| + | <br> | ||
| + | <p>We are confident that this construct can be a valuable addition to the iGEM registry and in future we hope that other iGEM teams take the challenge to solve the puzzle of its expression. As a fluorescent protein with an unusually long [https://en.wikipedia.org/wiki/Stokes_shift stoke-shift] it can be a useful tool in the crowded space occupied by the currently available reporter genes that are used by the worldwide family of iGEM teams.</p> | ||
===User Reviews=== | ===User Reviews=== | ||
Latest revision as of 16:15, 19 October 2016
Applications of BBa_K2060001
We developed the pBAD::mKeima construct as part of the Cardiff_Wales [http://2016.igem.org/Team:Cardiff_Wales/mkeima 'Fuel Project']. We aimed to induce expression from two strain of E.coli that either contained the previously characterised pBAD:LUXoperon construct or our own mKeima construct. Fluorescence by Unbound Excitation from Luminescence (FUEL) reaction describes a phenomonon whereby a light output can excite a unrelated fluorophore to produce light of a different wavelength. Therefore we aimed to induce a red-light output from cells expressing the mKeima protein when mixed with cells expressing the LUXoperon, which gives out blue light at approximately 480nm.
As described on our [http://2016.igem.org/Team:Cardiff_Wales/mkeima Wiki] we were able to induce blue light production from LUXoperon using a range of arabinose concentrations. However our efforts to demonstrate FUEL were thwarted by our inability to induce an output from cells containing the mKeima construct, which when excited at 440nm emits light at 620nm.
We attempted to induce pBAD:mKeima by growing bacteria at a range of temperatures and across a wide range of arabinose concentrations. Although our experimental control cells (which contained a pBAD:sfGFP construct) showed expression across these different parameters unfortunately we were unable to induce mKeima expression that resulted in a light output at 620nm.
Our attempts to induce expression of mKeima included experiments in which bacteria were grown under the following conditions:
1. Grow o/n without induction. Subculture (1/10) for 2hr. Induce with arabinose at concentrations between 100uM-10mM.
2. Grow o/n at 20C, 25C or 37C with arabinose induction at concentrations between 5uM-50mM.
3. Grow o/n without induction. Immediately induce with arabinose at concentrations between 100uM-5mM. Incubale cultures for 2-5h at 20C or 37C.
We are confident that this construct can be a valuable addition to the iGEM registry and in future we hope that other iGEM teams take the challenge to solve the puzzle of its expression. As a fluorescent protein with an unusually long stoke-shift it can be a useful tool in the crowded space occupied by the currently available reporter genes that are used by the worldwide family of iGEM teams.
User Reviews
UNIQ134592cc7a403660-partinfo-00000000-QINU UNIQ134592cc7a403660-partinfo-00000001-QINU