Team:Peking/Project/Luminesensor/Characterization

Orthogonality Test

It is our biggest concern whether LexA408-Luminesensor works independently of the endogenous LexA. If yes, maximizing its biological orthogonality will make the Luminesensor truly a plug-and-play device.

Two sections of testing expriments were carried out simultaneously. GFP was selected as a reporter and was fused downstream to two promoters (psulA408 and precA408) controlled by the Luminesensor with the 408 mutation and to two promoters (psulA and precA) controlled by the Luminesensor without the 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.

1. Experimental Design(Figure 4)
To prove the orthogonality, data and facts to prove that LexA408-VVD and the endogenous LexA work totally independently are needed. Considering the practical efficiency, two points of evidence were to be collected:

• 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 darkness.

Figure 4. Shows observable color of plates where cell cultures growing under totally dark or light conditions. Different colors are used to indicate three levels of GFP expression.

2. Detailed Methods
Experiments were carried out in parallel -- taking photos of the plates as visual evidence and measuring GFP to quantify the effect. Detailed protocols are as follows:

• 1. Visible display:
• • Inoculate a single colony into 1.5ml centifuge tube, shaking cultivate cells at 250 rpm/min for 2-3 h at 30℃, then the cell culture is ready for streaking;
  • Streak cell culture onto respective plates;
  • Incubate in total darkness and under light state at 30℃ for 1 or 2 days;
  • Take photos of correct plates;
• 2. Data collection:
• • Inoculate a proper single colony into liquid media;
  • Cultivate cells in a shaking state until platform stage at 30 ℃;
  • Dilute cell cultures with selective media at a ratio of 1:500;
  • Cultivate cells in a shaking state until the stationary stage at 30 ℃ in complete darkness or under blue light;
  • Harvest cells by centrifugation, then re-suspend pellets in PBS;
  • Measure GFP expression with 96-well-microplate reader;

3. Results
Below is a collage of our plates. The plates are placed in groups at 30℃ in either total darkness or under blue-light illumination. Visual results fit well with Figure 4. Visual appearance of the plates were positive evidence for us.

Figure 5(1). 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). Evidently there is no GFP expression, indicating that endogenous LexA proteins are capable of fully repress the expression of our wild type reporter gene, rendering our Luminesensor system no longer light-controllable.

Figure 5(2). Plate streaked with wild type E.coli transformed with GFP gene fused to the downstream of psulA408 (A) and precA408 (B). Evidently GFP is perfectly expressed, indicating endogenous LexA proteins are unable to bind to psulA408 and precA408, which suggested that the 408 promoter system is orthogonal to the endogenous SOS system.

Figure 5(3). Plate streaked with wild type E.coli transformed with mutated luminesensor and precA408-GFP or psulA408-GFP reporter gene 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.) Just as we predicted, the reporter gene is expressed in the dark and supressed by our luminesensor under light. This clearly proved that our 408 system is orthogonal to bacteria SOS system while retaining light-controllability.

Apart from those photos, more accurate results were obtained with Fluorescent Microplate Reader. Below are histograms based on our experimmental data. They clarify how fluoresence changes with lighting condition in different strains(Figure 6).

Figure 6(1). GFP expression level under control of precA408 promoter. Column 1 shows expression of precA-GFP reporter gene when transformed into wild-type E.coli without luminesensor. The high expression level indicates that the 408 reporter system is not interfered by endogenous LexA protein. The column 2 and column 3 are expression level of precA-GFP reporter gene co-transformed with our 408 form luminesensor into wild-type E.coli, and cultivated under light (column 2), or under light (column 3). The low expression level under light and hign expression level in the dark indicate that our 408 luminesensor system is totally light-controllable, and possessing a high dynamic range.

Figure 6(2). GFP expression level under control of psulA408 promoter. Every column shows data acquired under conditions same as its conterpart in Figure 7(1). The only difference is that the precA408 promoter has been replaced by psulA408 promoter.

It repeatedly turned out that:

1. reporters under wild-type promoters (psulA and precA) hardly expressed in either illuminated or dark state; their colonies are yellow. In contrast, GFPs fused to promoters psulA408 and precA408 were not blocked in strains, thus they looks bright green.

2. BL21 (DE3) strains expressing both the Luminesensor and its reporters shifted swiftly and sensitively from one states to the other, giving high definition output including lighting condition.

To sum up, LexA408-VVD-Luminesensor works independently of host genetic context.

Reference

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• 3. Roche, E.D., and Sauer., R.T.(2001). Identification of Endogenous SsrA-tagged Proteins Reveals Tagging at Positions Corresponding to Stop Codons. J. Biol. Chem., 276: 28509: 28515
• 4. Dimitrova,D., et al.(1997). A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli.Mol. Gen. Genet., 257: 205: 212
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