Difference between revisions of "Part:BBa K2060001:Experience"
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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.
 
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>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>
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<p>We attempted to induce pABD:mKeima by growing bacteria at a range of temperatures (20C, 25C, 37C) and across a wide range of arabinose concentrations (5uM-50mM). Although our experimental control cells (which contained a pBAD:sfGFP construct) showed expression across these different parameters we were unable to induce mKeima expression that resulted in a light output at 620nm.</p>
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<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>
 
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<p>The data below is taken from an experiment where <i>E.coli</i> were grown overnight at 20C at a wide range of arabinose concentrations. The light output was measured using a [http://diagnostics.tecan.com/products/reader_and_washer/microplate_readers/infinite_200_pro Tecan Infinite fluorometer]. The data shows that the sfGFP yielded the expected light output but that the pBAD:mKeima containing lines did not yield a light output. We did not test the accumlation of mKeima mRNA but our difficulties in visualising the correct light shows that this system requires further manipulation to yield the required output.</p>
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<p>Our attempts to induce expression of mKeima included experiments in which bacteria were grown under the following conditions:</p>
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<p>1. Grow o/n without induction. Subculture (1/10) for 2hr. Induce with arabinose at concentrations between 100uM-10mM.</p>
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<p>2. Grow o/n at 20C, 25C or 37C with arabinose induction at concentrations between 5uM-50mM.</p>
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<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>
  
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DATA HERE.
 
 
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<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 Stoke-Shift it can be a useful tool in the crowded space occupied by a range of fluorophores.</p>
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<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

UNIQcc24b586c55e05df-partinfo-00000000-QINU UNIQcc24b586c55e05df-partinfo-00000001-QINU