Difference between revisions of "Part:BBa K415021"

Latest revision as of 01:39, 27 October 2010

Pluxlac RBS-GFP TT PluxCI RBS-mCherry TT PtetR RBS-LuxR TT

This part displays red and green fluorescence under specific conditions. Fluorescence is activated by presence of both LuxR and 3OC6HSL. The LuxR is normally constitutively expressed, but can be repressed by TetR and derepressed by aTc. Green fluorescence is repressed by LacI and red fluorescence is repressed by CI.

Sequence and Features

Confirmation: BBa_K415021 was constructed by inserting Part:BBa_K182101 into BBa_K415013, which is Part:BBa_K415006 in Part:pSB3K3, a low to medium copy number plasmid. The following 1% agarose gel, prepared from agarose mix and 1% TAE and stained with SYBR® Safe DNA gel stain, presents confirmation of said construction. The part was run along the standard of Hyperladder 1. NcoI enzyme was used for the restriction mapping, resulting in bands of 414, 1256, and 4028 base pairs, which agrees with sequencing predictions. The part was also sequenced and confirmed in that way.

Figure 1

The following is the plasmid map of BBa_K415021 in backbone Part:pSB3K3.

Figure 2

The fluorescence of mCherry and GFP was then quantified using FACs(Fluorescence-activated cell sorting) running two samples: -AHL and +AHL. The samples were grown overnight in 4ml of LB with 3 mM IPTG with +/- 130uM AHL and Amp antibiotic in a 37C incubator in JM2.300 cells. Plasmid pRCV3(Plux-GFP) and pINV5(pLacIQ->lacI; pLac->GFP) were used as control to ensure adequate concentrations of AHL and IPTG induction. The fluorescence of mCherry and GFP were measured in arbitrary units.

Figure 3

The addition of AHL results in a 1385% increase in GFP fluorescence and 535% increase in mCherry fluorescence. The greater increase in fluorescence in the GFP may be attributed to the low degradation rate of the untagged GFP protein Part:BBa_E0040

The following are the FACs data sheets:

Figure 4: K415021 -AHL

Figure 5: K415021 +AHL