Difference between revisions of "Part:BBa K1897016"

Revision as of 12:50, 16 October 2016

LuxR + GFP

This part is made by 3A ligation (see: https://parts.igem.org/Help:Assembly/3A_Assembly) of LuxR BBa_K1897008 and green fluorescent protein (GFP) BBa_K1897014. LuxR is derived from Vibrio fischeri while GFP is derived from Aequeora victoria.

Usage and Biology

This part is intended to be used with the external addition of N-(3-oxo-hexanoyl)-homoserine lactone (AHL) in order to result in the expression of GFP.

The LuxR produced will bind to pLuxR promoter BBa_R0062 only in the presence of sufficient amounts of AHL. Normally in Vibrio fischeri, LuxI produces the AHL required. In this case however, since no LuxI is present in this part, external AHL has to be added in order to activate transcription of GFP. The GFP produced can then be detected when excited at 396 nm or 475 nm, resulting in fluroescence at 503 or 509 nm (Mishin et al., 2010).

Sequence and Features

Construction of LuxR+GFP

LuxR+GFP was made via 3A ligation of LuxR BBa_K1897008 and GFP BBa_K1897014. This was done by cutting GFP (BBa_K1897014) with EcoRI (at the Biobrick prefix) and SpeI (at the Biobrick suffix) and cutting LuxR (BBa_K1897008) with XbaI (at the Biobrick prefix) and PstI (at the Biobrick suffix). At the same time, the pSB1C3 backbone was cut with EcoRI and PstI. The 3 fragments were ligated together using T4 ligase, with a scar site formed between GFP and LuxR due to the compatible overhangs created by SpeI and XbaI. This resulted in an end product of a plasmid with GFP and LuxR joined together.

Verification of LuxR+GFP

-INSERT GEL PHOTO AS FIGURE 1-

Besides the verification of the band size via gel electrophoresis, we also verified the part by addition of external AHL. With no AHL added, fluorescence was very faint, and when 100 μm AHL was added, the fluorescence increased (Figure 2, Figure 3).

Figure 2: Induction of GFP production with/without AHL. Left Column: Green fluorescence microscopy images of LuxR+GFP bacteria of control with 0 μm AHL added (top) and with 100 μm AHL added (bottom). Right Column: Bright field fluorescence microscopy images of LuxR+GFP bacteria of control with 0 μm AHL added (top) and with 100 μm AHL added (bottom).

GFP levels were also measured using a microplate reader in the presence or absence of AHL, and the results obtained over a period of 6 hours can be seen in Figure 3. The level of fluorescence is higher for induced (100 μm AHL) than non induced (0 μm AHL), indicating that the addition of AHL induces expression of GFP from pLuxR.

Figure 3: GFP quantifications via microplate reader of LuxR+GFP for 6 hours is shown in the presence of 0 or 100 μm AHL incubation. The level of fluorescence is higher for induced (100 μm AHL) than non induced (0 μm AHL), indicating that the addition of AHL induces expression of GFP from pLuxR.