• Introduction
• Objective
• Method
  • -Design
  • -Parts construction
• -Evaluation
  • Result
• Future work

Introduction

Right sensor

1. YF1/ FixJ

YF1/ FixJ system is blue light (480 nm) sensing system. YF1 is a fusion protein, heme-binding PAS sensor domain of FixL from Bradyrhizobium japonicum(FixL) and the LOV blue light sensor domain of Bacillus subtilis YtvA(YtvA). The Histidine kinase YF1 employs a light-oxygen-voltage, blue light photosensor domain. FixJ is YF1’s cognate response regulator. In the absence of blue light, YF1 phosphorylates FixJ, and phosphorylated FixJ drives robust gene expression from the FixK2 promoter(Ref. 1,2,3).

2. Rhodopsin
　 Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis (N. pharaonis) show phototaxis by responding to changes in light color and intensity using receptors called sensory rhodopsin I and II (SRI and SRII). The SR proteins are seven-transmembrane retinylidene photoreceptors, which transmits blue light signal (λmax 487 nm) to their corresponding transducers HtrI and HtrII respectively. signals to Htr proteins via helix-helix interaction. Htr protein consists of two transmembrane helices and a cytoplasmic methyl-accepting and His-Kinase domain, and belongs to histidine kinase / phosphoreregulator two-component system for regulating cells’ flagellar motors for phototaxis (Ref. 4,5).


Objective
　Reguration of taRNA expression by light sensor protein



Method
-Design
We construct HHRs containing RBS is downstream of the Pconst promoter (low), and taRNA which binds HHR and inactivates HHR’s self-cleaving activity is on downstream of PompC or Pfixk2 promoter. Under dark condition, taRNA is expressed and inactivates HHR’s self-cleaving activity. On the other hand, under blue light condition, taRNA isn’t expressed and HHR self-cleaves. Becouse of HHR self-cleaving, GFP’s RBS is exposed and GFP is expressed.

-Parts construction
1. YF1/ FixJ
1.) BioBrick part BBa_K1053210 (Tokyo-NoKoGen2013) is fused with HHR connecting with GFP (BBa_K1053004(Tokyo-NoKoGen2013)) or HHR* connecting with GFP (BBa_K1053005(Tokyo-NoKoGen2013)) by using Overlap PCR.
2.) The PCR products were gel purified and digested with XbaⅠ and PstⅠ. The digested products were ligated into pSB1A3 vector.(Fig. 1)
3.) Constructed plasmids were transformed into E.coli DH5α.

Fig. 1 pSB1A3 – Pconst. – YF1/FixJ - Pfixk2 – TR(12)- HHR - GFP - DT




Fig. 2 pSB1A3- Pconst. – YF1/FixJ - Pfixk2 – TR(12)- HHR - GFP - DT



2. Rhodopsin
1.) BioBrick part BBa_K769003 (Tokyo-NoKoGen2012) which consists of a chimeric sensory rhodopsin and its cognate transducer from N. pharaonis and the histidine kinase domain of EnvZ from E. coli, that is fused with HHR connecting with GFP (BBa_K1053004(Tokyo-NoKoGen2013)) or HHR* connecting with GFP (BBa_K1053005(Tokyo-NoKoGen2013)) by using Overlap PCR.
2.) The PCR products were gel purified and digested with EcoRⅠ and PstⅠ. The digested products were ligated into pSB1A3 vector.(Fig. 3) 3.) Constructed plasmids were transformed into E.coli DH5α.

Fig.3 pSB1A3 - Pconst. - SRⅡ- HtrⅡ- EnvZ - PompC - HHR - GFP - DT




Fig.4 pSB1A3 - Pconst. - SRⅡ- HtrⅡ- EnvZ - PompC - HHR* - GFP - DT





-Evaluation
YF1/ FixJ
1.) Construct made in –Parts construction- was used to transform into E.coli DH5α.
2.) The transformants were pre-cultured in 3 mL LB medium overnight at 37 degrees celsius, under dark condition.
3.) 450 μL of pre-cultures were inoculated into 3 mL LB medium inside and incubated either under dark or blue light conditions. OD595 and GFP fluorescence intensity were measured at certain time.(Fig. 1)



Result
There is no clear difference of light and dark condition of HHR-GFPuv or HHR*-GFPuv.
The cause of this result is that excitation light of GFP and YF is almost the same.

Result 1 Evaluation of transformed E.coli under the light or dark condition.

We evaluate Pconst. – taR12- Pconst. (low) – YF1/ FixJ – Pfixk2 – HHR(Fig. 6).


Fig. 6 Pconst. – taR12- Pconst. (low) – YF1/ FixJ – Pfixk2 - HHR


We estimate in light GFP is expressed by HHR’s self cleavage and in dark GFP isn’t expressed by taRNA’s expression. If HHR’s active site is mutated, GFP isn’t expressed.
The GFP fluorescence intensity was taken after main culture for 12 h by Plate Reader in this evaluation method.



Result 2 Evaluation of transformed E. coli under the light or dark condition.


If HHR’s active site isn’t mutated difference between in light or dark is observed. This result is suggest that in light taRNA is expressed and HHR’s auto cleavage is inhibited.
RNA oscillator’s result is difference between taRNA’s existence or absence isn’t observed. We think that this difference is caused by difference between Pconst.(low) and Pbad.




Future Work
We want to evaluate Pconst. - Yf1/FixJ - Pfixk2- TR(12)- HHR- GFP- DT and Pfixk2 - taR12 - Pconst.(low) TR(12)- HHR- GFP- Pconst.- YF1/ FixJ by changing main culture time and changing evaluation method again.
We want to synchronize oscillation cycle with Twinkle.coli by light sensor protein in the future.



Reference
[1] J. Mol. Biol.(2009) 385, 1433-1444
[2] J. Mol. Biol. (2012) 416, 534-542
[3] Bacteriology (1998) 180, 5251-5255
[4] Xue-Nong Zhang et al. (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA
[5] Wouter D. Hoff et al. (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct.

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