Team:Chiba/Experiments:LuxR mutant

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Revision as of 03:28, 30 October 2008

LuxR mutant (Under Planning)

Design

Probably, the most straightforward approach to make variations in delay time is to create a number of LuxR mutants with different sensitivity. We thought the higher the sensitivity is, the shorter the delay time would be. The lower the sensitivity is, the slower the switch response should be.

At least, two papers on LuxR mutants ^(1),(2) are available. Among the many candidates, we chose the following three mutations;

1. Ile45Phe: This mutation make LuxR 10x more sensitive to Lux-type AHL⁽¹⁾
2. Leu42Ala: Sensitivity (to AHL) becomes 1/15 of the wildtype LuxR⁽²⁾
3. Leu42Ser: Sensitivity (to AHL) becomes 1/1,000 of the wildtype LuxR⁽²⁾

Fig. 3 I45F mutation

Fig. 4 L42A,S mutation

Fig.3, Fig.4 Hypothetical positions of residues in TraR corresponding to those found to modulate acyl-HSL specificity in LuxR.　The crystal　structure of the LuxR homologue TraR (PDB 1L3L) has been determined(3).

Experiment

Mutant construction:
Using sewing PCR, we introduced each of three above mutations into LuxR. Some of the parts are await for sequence verification.

Test communication:

1. Transformed sender (Ptet-luxI), mutant LuxR Receiver (Ptet-mLuxR-Plux-GFP) and WT LuxR Receiver into E. coli strains (JW1908)
2. Separately inoculated Sender, WT Receiver (wild type luxR/BW⊿FliC) and mutated Receiver (1point mutation/BW⊿FliC) in liquid media for 12 h at 37℃.
3. Inoculated again in liquid media upto about OD600=2 at 37℃
4. Washed, adjusted the cell density, and mixed them. (Sender:Receiver=1000μL:1000μL)
5. Incubated at 30°C, measuring the fluorescent intensity intermittently

Status (Ongoing)

1. We have constructed all three mutants described above.
2. We haven't seen any noticeable difference in response-time between wt-luxR and I45F (hyper-sensitive) mutant of LuxR. Sucks big time. We think this is probably due to that LuxR is already sensitive enough.
3. We have just constructed Leu42 mutants, but they are waiting for the sequence verification (as of Oct 29th). We will conduct the communication experiment as soon as sequencing get completed. Hope we will see the significant time-delay, ideally without any decrease in the endpoint signal intensity.

One more plan; Searching for Delay Switcher

We are planning to do a simple directed evolution to isolate the 'delay switcher' mutant of LuxR.

Reference

1. Collins CH et.al. Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. Mol. Microbiol. 55, 712–723 (2005)
2. Koch B et.al.:The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors. Microbiol. 2005 Nov;151(Pt 11):3589-602.
3. Zhang RG et. al. Structure of a bacterial quorum-sensing transcription factor complexed with pheromone and DNA., Nature, 417, 971-974 (2002)

Table 1 Seisitivity of LuxR mutants. Data modified from Collins et.al. Mol. Microbiol. 55, 712–723 (2005)

Table 2 Ahl activation of mutagenized LuxR molecules. Data modfied from B.Kock et al. Microbiology (2005), 151, 3589-3602

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