Difference between revisions of "Part:BBa K2447000"

Revision as of 14:54, 28 October 2017

Extracellular phosphate sensor with GFP reporter

Figure 1: PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP

Improvement over previous iGEM part BBa_K116404 (NYMU Taipei 2008)

The two parts (Bba_K2447000 & BBa_K116404) listed below are inserted into pBbE2k backbone and subsequently characterized in E.coli MG 1655. These cells were grown in LB medium before transferring into MOPS medium (a minimal nutrient medium) for characterization with a micro-plate reader. Varying concentrations of phosphate ions from 0 to 1000 uM were added and GFP expression was monitored.

By replacing the weaker RBS BBa_B0032 of the original part with a stronger RBS BBa_B0034, we have successfully constructed an improved phosphate sensor-GFP reporter. Our part shows, on average, 40 fold increase in GFP expression (Figure 3) when compared to the previous version of the construct (http://2008.igem.org/Team:NYMU-Taipei/Project/Phosphate). The original phosphate construct is also insensitive to high phosphate concentrations above 50 µM where similar levels of GFP expression are observed (Figure 4). Unlike the previous construct, our improved phosphate construct is much more sensitive to various phosphate concentrations from 0 to 1000 µM, particularly at phosphate concentrations above 50 µM.

Figure 2: Side by side comparison of our improved construct Bba_K2447000 & the original construct BBa_K116404. Our proposed part exhibited greater GFP expression at every phosphate concentration and exhibited much higher sensitivity to phosphate levels above 50uM.

Figure 3: Our improved phosphate construct is much more sensitive to phosphate concentrations above 50 uM unlike the original construct.

Figure 4: Original phosphate construct designed by the Taiwanese team.

Sequence and Features