Part:BBa_K819005:Characterization

Constitutive LuxBrick Generator

Summary

Design

Characterization

Reference

This page shows the experimental characterization of this luminesensor. All the results show that the luminesensor works quite well, achieving high sensitivity, bio-orthogonality, high on/off ratio, and independence of exogenous chromophore at the same time.

(For more detail information please visit our [http://2012.igem.org/Team:Peking/Project/Luminesensor/Characterization wiki])

Sensitivity

We tested the sensitivity of Luminesensor by examine the light-dependent transcriptional activity of a GFP-ssrA reporter. ssrA is a protein tag that induces fast degradation of protein, which in our case facilitated the observation of transcriptional activity. Based on the consideration of guaranteeing accuracy and precision, our setup (Figure 1):consists of three central parts: light source, incubator and 48-well plate.

FIG.1 The set-up of sensitivity tests.

On account of high sensitivity, protecting the system from the preventable light exposure with the purpose of acquiring the accurate results which is the true reflection of our sensitivity is necessary. In order to solve the problems, we focus on two foremost aspects: utilizing attenuators to weaken the light intensity and using tin foil to avoid light leakage. For more details about how we conducting the experiment, see experiment procedures. In our experiments, illumination with different light intensity conditions at 460nm peak light from blue LED arrays for 16 hours show marked light-depressed reporter gene transcription, which indicates that under different blue light exposure conditions, there was hardly any light-induced reporter gene transcriptional activity. But when in the dark environment (packaged with three layers of aluminum foil), our systems showed extremely high GFP expression (Figure 2):

FIG.2 The sensitivity of Luminesensor.

Orthogonality Test

It is our biggest concern whether lexA408 Luminesensor works independently of endogenous LexA. If yes, maximizing its biological orthogonality will make Luminesensor a really plug-and-play device.

Two sections of testing expriments were carried out simultaneously. GFP was selected as a reporter and was fused downstream to 2 promoters (psulA408 and precA408) controlled by Luminesensor with 408 mutation and to 2 promoters (psulA and precA) controlled by Luminesensor without mutation. GFP expression is expected to have a negative relation with repression activity. To be more specific, higher level of green fluorescent indicates weaker repression effect; lower expression of GFP stands for stronger repression.

To prove the orthogonality, facts that LexA408-VVD and endogenous LexA work totally independently are needed. Considering practical efficiency, 2 points of evidence are to collect:
1. Promoters psulA and precA are repressed in wild-type Ecoli strains while promoters psulA408 and precA408 are not blocked in wild-type strains;
2. LexA408-VVD Luminesensor efficiently represses its target under blue illumination, while it does not repress targets in total dark.

FIG.3 The design of orthogonality tests.

Below is a collage of our plates. The plates are placed in groups at 30℃ in either total dark or blue illumination. Visual results fit well with Figure 3. Visual look of plates were positive evidence for us.

FIG.4 Plate streaked with wild type E.coli transformed with wild type luminesensor and GFP fused to LexA responsive promoters, and cultivated in the dark (A), or under light (B).

FIG.5 Plate streaked with wild type E.coli transformed with GFP gene fused to the downstream of psulA408 (A) and precA408 (B).

FIG.6 Plate streaked with wild type E.coli transformed with mutated luminesensor and GFP gene fused to the downstream of precA408 and psulA408 and cultivated in the dark or under light.(A: luminesensor + precA408-GFP, cultivated in the dark. B: luminesensor + precA408-GFP, cultivated under light. C: luminesensor + psulA408-GFP, cultivated in the dark. D: luminesensor + psulA408-GFP, cultivated under light.)

Improving Dynamic Performance

To test whether our designated mutations would improve the dynamic performance of Luminesensor in respect to reversibility and the on/off ratio, we co-transformed the psulA-GFP (GFP driven by luminesensor repressible promoter psulA) plasmid with four versions of Luminesensor plasmid into BL21 (ΔLexA ΔSulA): the original LexA-VVD(WT), LexA-VVD(I74V), LexA-VVD(M135I), and LexA-VVD(I74V+M135I). Resulted four strains were designated as LV-WT, LV-74, LV-135, LV-74-135, respectively. The overnight culture of the four strains were diluted 500 times and divided into two groups: one exposed to blue light and the completely wrapped with aluminum foil. After incubation for 16 hours, GFP expression levels were measured. As shown in figure below, LV-135 has an increased on/off ratio compared to the original LexA-VVD, while the LV-74 and LV-74-135 show reduced on/off ratios, which is in accordance with our modeling result.

FIG.7 Effects of introduced mutations on the performance (on/off ratio) of Luminesensor.

In order to demonstrate the reversibility of our Luminesensor, we recorded the different target gene expression in different dark time using RFP as the reporter. The expression of RFP increases with the increase of dark time.

FIG.8 The time course of RFP expression controlled by LV-WT.

In order to determine whether the LexA-VVD(M135I) Luminesensor has enhanced reversibility in comparison to the original LexA-VVD, the temporal change of GFP expression level of dilated overnight culture of the strains LV-WT and LV-135 under blue light were measured at 2 hour intervals for 26 hours. As shown in figure below, the GFP expression level of both of the two strains began to rise after incubating at dark for about 10 hour. We speculated that the GFP expression level of the two strains is mainly determined by the equilibrium between GFP production and degradation and the dimerized LexA-VVD(WT) and LexA-VVD(135) dissociate in a shorter time scale compared to the time needed to establish the equilibrium of the GFP production and degradation.

FIG.9 The time course of GFP expression controlled by LV-WT and LV-135.