Latest revision as of 09:42, 16 October 2014

lux pL controlled luxR with lux pR controlled gfp (LVA-tag) Lux pL controlled LuxR + lux pR autoinducing LuxI (lva tag) + lux pR controlled GFP(lva tag).

Description

The lux pL controlled luxR with lux pR is known as quorum sensing promoter. the lux pR is at slightly activated under normal circumstance and produces LuxI protein that synthesize a kind of acyl-homoserine lactone (AHL), which is a small molecule that can diffuse across the cell membrane and mediate intercellular coupling when it reaches the threshold as enough biomass accumulated. AHL will bind intracellular protein LuxR, which is also consecutively produced by luxR gene. The LuxR-AHL complex can activate the luxI promoter, and the positive feedback loop is built. That means once the biomass of bacteria reach the threshold, not only the expression of luxI protein gene will be increased but also that of the GFP downstream of the same quorum sensing promoter.

The GFP molecule acts as a photosensitizer, releasing free radicals upon exposure that produce reactive oxygen species (ROS) including H₂O₂. At the peak of oscillation, considerable vapour-phase H₂O₂ is produced by exposing GFP containing cells to fluorescent light. Conversely, at the trough of oscillation, cells contain almost no GFP, and therefore produce very little H₂O₂ upon fluorescing. Bursts of light thus generate bursts of H₂O₂ vapour whose concentration depends on the oscillating GFP level, just as periodic production of NDH-2 did previously. Indeed, this strategy was similarly able to synchronize our sensor array. Numerous controls were performed to ensure that synchronized oscillations did not occur at low fluorescence intensities.

Experimental Data

Fig.1 showed the correct result of this part construction and Fig.2 showed the distinguish of BBa_K1036002 which was without lux promoter and BBa_K1036003 which was under lux promoter control.
Fig.3 showed the SDS-PAGE of pSB1C3-gfp-luxI expression and the band about 35 kDa was confirmed as GFP and the band about 25 kDa was LuxI according to the MALDI-TOF-MS data.

Supplementary Information

A informational contribution from iGEM14_XMU-China.
Group: iGEM14_XMU-China
Author: Tang Chun
Summary: iGEM14_XMU-China makes a reasonable explanation for the misfolding GFP by this devices.

As the SDS-PAGE shows(Figure 1), a large amount of GFP-LVA and LuxI-LVA appear in pellet where misfolding proteins often exist. Both proteins directly affect the oscillation result. And it is critical to find out the reason for misfolding proteins.

Figure 1. SDS-PAGE analysis of E.coli K strain (DH5α). (a) Lane 1-2: supernatant and pellet of original DH5α; Lane 3-4: supernatant and pellet of strain with single plasmid A1 (BBa_K1036003); Lane 5-6: supernatant and pellet of strain with both plasmids A1 (BBa_k1036003) and B (BBa_K1036000). The red arrows indicate the misfolding GFP-LVA protein (27.6 kDa) in the precipitation. (b) Lane 1-2: supernatant and pellet of original BL21; Lane 3-4: supernatant and pellet of strain with single plasmid A1 (BBa_K1036003); Lane 5-6: supernatant and pellet of strain with both plasmids A1 (BBa_K1036003) and B (BBa_K1036000). The blue arrows indicate LuxR (27.5 kDa), GFP-LVA (27.6 kDa) and AiiA-LVA (28.7 kDa) in the supernatant. The orange arrows indicate LuxI-LVA (22.4 kDa) in the supernatant. (The marker of b was not in right position, however, the proteins were confirmed by MALDI-TOF-TOF .)

The 2012 published paper[1] reveals an unexpected behavior of Lux pR (BBa_R0062). In the absence of autoinducer 3OC6 (AHL), LuxR binds to Plux (Lux pR) and activates backwards transcription (Figure 2).

Figure 2. Relative RFP fluorescence for a control construct designed to measure backwards transcription from Lux pR. Addition of LuxR and 3OC690 (AHL) as indicated. Error bars in all panels are one standard deviation.

With this device, when LuxR activates the backward transcription, RNA polymerase will be blocked by the terminators B0015. Actually, the reverse terminated efficiency of B0015 is 0.295(CC)[2] which may lead to leakage transcription. However, the correct sequence of GFP-LAA can’t be transcribed during the backwards transcription, even if the minus-strand of GFP-LAA could be transcribed, the sequence of the RNA is not in the right direction of GFP-LAA, hence incorrect amino acid sequences may be translated, resulting in misfolding GFP just as the SDS-PAGE shows (Figure 1).
iGEM14 XMU-China involved sequence comparison to investigate the difference between the original and the registry parts. We find that the original Lux pR has 20bp overlapping sequence with original Lux pL. There is a restriction enzyme cutting site (EcoR I) at the 56bp of original Lux pR (Figure 3).
Parts registry truncate the original Lux pR at 56bp to get the 55bp Lux pR (BBa_R0062). On the contrary, Lux pL (BBa_R0063) is longer the original Lux pL, and at the end of BBa_R0063 is initial part of 41bp LuxR (BBa_C0062). Thus new problems arise—is the modification of original QS promoter reasonable? Does the modification result in the unexpected backward transcription?

Figure 3. Schematic of original QS promoter.

Quorum sensing system is so widely used in the synthetic biology, we think it’s remarkable to make it clear. We highlight the abnormal phenomenon of QS oscillation which may be caused by imperfect simplification for the very first time. We hope that more efforts could be made to figure out the interaction between QS oscillation parts.[3]

Reference
[1] Eckdahl T, Sawyer E M, Barta C, et al. Bacterial logic devices reveal unexpected behavior of frameshift suppressor tRNAs[J]. Interdisciplinary Bio Central, 2012, 4(1): 10.
[2] BBa_B0015
[3] Danino T, Mondragón-Palomino O, Tsimring L, et al. A synchronized quorum of genetic clocks[J]. Nature, 2010, 463(7279): 326-330.