Difference between revisions of "Team:NUS Singapore/Improvement"

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<h1>Part Improvement</h1>
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    <p>Our team has decided to improve upon the <a href="http://parts.igem.org/Part:BBa_K116404">BBa_K116404</a> phosphate sensor-GFP reporter which was constructed by NYMU Taipei in 2008. The part functions as an external phosphate ion sensor, where high phosphate concentration is present, the phosphate promoter pPhoB would be repressed and stopping downstream GFP production. </p>
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    <p>By replacing the weaker RBS 32 of the original part with a stronger affinity RBS 34, we have successfully constructed <a href="http://parts.igem.org/Part:BBa_K2447000">BBa_K2447000</a> phosphate sensor-GFP reporter. Our part is much more sensitive to phosphate concentrations even at phosphate concentrations of 40uM and above. </p>
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    <h3>Characterisation Protocol: </h3>
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    <p>Transformed MG 1655 cells are incubated in LB+K broth at 37 degree for 24 hours before being refreshed to reach an OD of 0.1. Cells are washed in MOPS medium (0.2% glucose) and subsequently re-suspended in MOPS. Next, cells are loaded onto 96 well plate preloaded with various concentrations of phosphate concentrations. 10 mins interval reading of OD 600 and GFP absorbance was conducted over a continuous 8 hours run of the microplate reader at 37 degree. </p>
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    <p>Figure 1: BBa_K2447000 Phosphate sensor coupled to GFP reporter. Strong GFP expression was elucidated for phosphate concentrations above 50uM.</p>
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Revision as of 08:43, 20 October 2017

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Part Improvement

Our team has decided to improve upon the BBa_K116404 phosphate sensor-GFP reporter which was constructed by NYMU Taipei in 2008. The part functions as an external phosphate ion sensor, where high phosphate concentration is present, the phosphate promoter pPhoB would be repressed and stopping downstream GFP production.

By replacing the weaker RBS 32 of the original part with a stronger affinity RBS 34, we have successfully constructed BBa_K2447000 phosphate sensor-GFP reporter. Our part is much more sensitive to phosphate concentrations even at phosphate concentrations of 40uM and above.

Characterisation Protocol:

Transformed MG 1655 cells are incubated in LB+K broth at 37 degree for 24 hours before being refreshed to reach an OD of 0.1. Cells are washed in MOPS medium (0.2% glucose) and subsequently re-suspended in MOPS. Next, cells are loaded onto 96 well plate preloaded with various concentrations of phosphate concentrations. 10 mins interval reading of OD 600 and GFP absorbance was conducted over a continuous 8 hours run of the microplate reader at 37 degree.

Figure 1: BBa_K2447000 Phosphate sensor coupled to GFP reporter. Strong GFP expression was elucidated for phosphate concentrations above 50uM.