Team:Tokyo-NoKoGen/light

From 2013.igem.org

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YF1/ FixJ system is blue light (480 nm) sensing system. YF1 is a fusion protein, heme-binding PAS sensor domain of FixL from <I>Bradyrhizobium japonicum</I>(FixL) and the LOV blue light sensor domain of <I>Bacillus subtilis</I> 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 F<sub>ixK2</sub> promoter(Ref. 1,2,3).
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YF1/ FixJ system is blue light (480 nm) sensing system. YF1 is a fusion protein, heme-binding PAS sensor domain of FixL from <I>Bradyrhizobium japonicum</I>(FixL) and the LOV blue light sensor domain of <I>Bacillus subtilis</I> 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).
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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).
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Halophilic archaea, such as <I>Halobacterium salinarum</I> and<I> Natronobacterium pharaonis (N. pharaonis)</I> 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).
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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. <BR>
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We construct HHRs containing RBS is downstream of the P<sub>const</sub> promoter (low), and taRNA which binds HHR and inactivates HHR’s self-cleaving activity is on downstream of P<sub>ompC</sub> or P<sub>fixk2</sub> 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. <BR>
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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. <BR>
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. <BR>
2.) The PCR products were gel purified and digested with XbaⅠ and PstⅠ. The digested products were ligated into pSB1A3 vector.(Fig. 1)<BR>
2.) The PCR products were gel purified and digested with XbaⅠ and PstⅠ. The digested products were ligated into pSB1A3 vector.(Fig. 1)<BR>
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3.) Constructed plasmids were transformed into E.coli DH5α.
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3.) Constructed plasmids were transformed into <I>E.coli</I> DH5α.
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</P>
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<img src=https://static.igem.org/mediawiki/2013/2/29/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A33.PNG>
<img src=https://static.igem.org/mediawiki/2013/2/29/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A33.PNG>
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<BR>Fig. 1 pSB1A3 – Pconst. – YF1/FixJ - Pfixk2 – TR(12)- HHR - GFP - DT<BR><BR><BR><BR>
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<BR>Fig. 1 pSB1A3 – P<sub>const.</sub> – YF1/FixJ - P<sub>fixk2</sub> – TR(12)- HHR - GFP - DT<BR><BR><BR><BR>
<img src=https://static.igem.org/mediawiki/2013/5/51/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A34.PNG><BR>
<img src=https://static.igem.org/mediawiki/2013/5/51/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A34.PNG><BR>
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Fig. 2  pSB1A3- Pconst. – YF1/FixJ - Pfixk2 – TR(12)- HHR - GFP - DT
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Fig. 2  pSB1A3- P<sub>const.</sub> – YF1/FixJ - P<sub>fixk2</sub> – TR(12)- HHR - GFP - DT
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2. Rhodopsin<BR>
2. Rhodopsin<BR>
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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. <BR>
+
1.) BioBrick part BBa_K769003 (Tokyo-NoKoGen2012) which consists of a chimeric sensory rhodopsin and its cognate transducer from <I>N. pharaonis</I> and the histidine kinase domain of EnvZ from <I>E. coli</I>, 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. <BR>
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2.) The PCR products were gel purified and digested with EcoRⅠ and PstⅠ. The digested products were ligated into pSB1A3 vector.(Fig. 3)
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2.) The PCR products were gel purified and digested with <I>Eco</I>RⅠ and <I>Pst</I>Ⅰ. The digested products were ligated into pSB1A3 vector.(Fig. 3)
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3.) Constructed plasmids were transformed into E.coli DH5α.<BR>
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3.) Constructed plasmids were transformed into <I>E.coli</I> DH5α.<BR>
<img src=https://static.igem.org/mediawiki/2013/e/e0/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A37.PNG><BR>
<img src=https://static.igem.org/mediawiki/2013/e/e0/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A37.PNG><BR>
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Fig.3  pSB1A3 - Pconst. - SRⅡ- HtrⅡ- EnvZ - PompC - HHR - GFP - DT<BR><BR><BR><BR>
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Fig.3  pSB1A3 - P<sub>const.</sub> - SRⅡ- HtrⅡ- EnvZ - P<sub>ompC</sub> - HHR - GFP - DT<BR><BR><BR><BR>
<img src=https://static.igem.org/mediawiki/2013/3/35/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A38.PNG><BR>
<img src=https://static.igem.org/mediawiki/2013/3/35/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A38.PNG><BR>
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Fig.4  pSB1A3 - Pconst. - SRⅡ- HtrⅡ- EnvZ - PompC - HHR* - GFP - DT<BR><BR><BR><BR>
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Fig.4  pSB1A3 - P<sub>const.</sub> - SRⅡ- HtrⅡ- EnvZ - P<sub>ompC</sub> - HHR* - GFP - DT<BR><BR><BR><BR>
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YF1/ FixJ<BR>
YF1/ FixJ<BR>
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1.) Construct made in –Parts construction- was used to transform into E.coli DH5α.<BR>
+
1.) Construct made in –Parts construction- was used to transform into <I>E.coli</I> DH5α.<BR>
2.) The transformants were pre-cultured in 3 mL LB medium overnight at 37 degrees celsius, under dark condition.<BR>
2.) The transformants were pre-cultured in 3 mL LB medium overnight at 37 degrees celsius, under dark condition.<BR>
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)<BR>
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)<BR>
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4.) We evaluated Pconst. – taR12- Pconst. (low) – YF1/ FixJ – Pfixk2 – HHR(Fig. 6).<BR><BR>
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4.) We evaluated P<sub>const.</sub> – taR12- P<sub>const.</sub> (low) – YF1/ FixJ – P<sub>fixk2</sub> – HHR(Fig. 6).<BR><BR>
<img src=https://static.igem.org/mediawiki/2013/3/37/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A313.PNG><BR>
<img src=https://static.igem.org/mediawiki/2013/3/37/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A313.PNG><BR>
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Fig. 6  Pconst. – taR12- Pconst. (low) – YF1/ FixJ – Pfixk2 - HHR<BR><BR>
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Fig. 6  P<sub>const.</sub> – taR12- P<sub>const.</sub> (low) – YF1/ FixJ – P<sub>fixk2</sub> - HHR<BR><BR>
<p style="line-height:110%">
<p style="line-height:110%">
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.
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.
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There is no clear difference of light and dark condition of HHR-GFPuv or HHR*-GFPuv.<BR>
There is no clear difference of light and dark condition of HHR-GFPuv or HHR*-GFPuv.<BR>
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The cause of this result is that excitation light of GFP and YF is almost the same.<BR>
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The cause of this result is that excitation light of GFP and YFⅠ is almost the same.<BR>
</p>
</p>
<img src=https://static.igem.org/mediawiki/2013/b/b5/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A39.PNG>
<img src=https://static.igem.org/mediawiki/2013/b/b5/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A39.PNG>
<BR>
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Result 1  Evaluation of transformed E.coli under the light or dark condition.
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Result 1  Evaluation of transformed <I>E.coli</I> under the light or dark condition.
<BR><BR>
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<p style="line-height:110%">
<p style="line-height:110%">
  If HHR’s active site isn’t mutated difference between in light or dark is observed. This result is suggested that in light taRNA is expressed and HHR’s auto cleavage is inhibited.<BR>
  If HHR’s active site isn’t mutated difference between in light or dark is observed. This result is suggested that in light taRNA is expressed and HHR’s auto cleavage is inhibited.<BR>
-
  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.<BR>
+
  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 P<sub>const.</sub>(low) and P<sub>bad.</sub><BR>
</P>
</P>
<img src=https://static.igem.org/mediawiki/2013/c/c8/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A310.PNG><BR>
<img src=https://static.igem.org/mediawiki/2013/c/c8/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A310.PNG><BR>
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Result 2  Evaluation of transformed E. coli under the light or dark condition.
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Result 2  Evaluation of transformed <I>E. coli</I> under the light or dark condition.
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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.<BR>
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We want to evaluate P<sub>const.</sub> - Yf1/FixJ - P<sub>fixk2</sub>- TR(12)- HHR- GFP- DT and P<sub>fixk2</sub> - taR12 - P<sub>const.</sub>(low) TR(12)- HHR- GFP- P<sub>const.</sub>- YF1/ FixJ by changing main culture time and changing evaluation method again.<BR>
We want to synchronize oscillation cycle with Twinkle.coli by light sensor protein in the future.<BR>
We want to synchronize oscillation cycle with Twinkle.coli by light sensor protein in the future.<BR>
</p>
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Reference<BR>
Reference<BR>
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[1] J. Mol. Biol.(2009) 385, 1433-1444<BR>
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[1] <I>J. Mol. Biol.</I>(2009) 385, 1433-1444<BR>
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[2] J. Mol. Biol. (2012) 416, 534-542<BR>
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[2] <I>J. Mol. Biol. </I>(2012) 416, 534-542<BR>
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[3] Bacteriology (1998) 180, 5251-5255<BR>
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[3] <I>Bacteriology </I>(1998) 180, 5251-5255<BR>
-
[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 <BR>
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[4] Xue-Nong Zhang <I>et al.</I> (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA <BR>
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[5] Wouter D. Hoff et al. (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct.<BR>
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[5] Wouter D. Hoff <I>et al.</I> (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct.<BR>
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Revision as of 21:17, 27 September 2013

Team:Tokyo-NoKoGen - 2013.igem.org


Right sensor




Introduction


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)
4.) We evaluated 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

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 YFⅠ is almost the same.


Result 1 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 suggested 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.


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






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.