Difference between revisions of "Part:BBa K343003"

 
(24 intermediate revisions by 3 users not shown)
Line 1: Line 1:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K343003 short</partinfo>
 
<partinfo>BBa_K343003 short</partinfo>
 
+
<p style="text-align: justify;">
Sensory Rhodopsin II bluelight receptor fused to its transducer, HtrII, with a 27 BP linker region.  This is fused to Salmonella enterica serovar typhimurium chemotaxis protein Tar, so that the protein effectively couples the input from the receptor to the chemotaxis pathway and reduces the amount of phosphorylated CheY, which results in a lowered tumbling frequency.
+
<br>
The fusion between HtrII and Tsr is an M-fusion in the HAMP domain, which is supposed to give it maximum activity.
+
Sensory Rhodopsin II bluelight receptor fused to its transducer, HtrII, with a 27 BP linker region.  This is fused to ''Salmonella enterica serovar typhimurium'' chemotaxis protein Tar, so that the protein effectively couples the input from the receptor to the chemotaxis pathway and reduces the amount of phosphorylated CheY, which results in a lowered tumbling frequency.
 +
The fusion between HtrII and Tsr is an M-fusion in the HAMP domain, which is supposed to give it maximum activity. M-fusion emans that the last residue in the HtrII part of the fusion in the HAMP domain, consists of the aminoacid methionine. The other fusions which have been constructed are P- and G-fusions, named after the same principle. [https://parts.igem.org/Part:BBa_K343003#References [1]]
 
   
 
   
Sequencing confirmed that the sequence is identical to the found in the article by Jung, Spudich E, Trivedi and Spudich J in the article: An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli. (1)
+
Sequencing confirmed that the sequence is identical to that found in the article by Jung, Spudich E, Trivedi and Spudich J in the article: An Archaeal Photosignal-Transducing Module Mediates Phototaxis in ''Escherichia coli''. [https://parts.igem.org/Part:BBa_K343003#References [1]]
 +
<br>
 +
</p>
  
===Background===
+
==Background==
 
====The mechanism of bacterial motility====
 
====The mechanism of bacterial motility====
Phototaxis is naturally not present in E.Coli, so that the wildtype does not change it's motility in response to light. The bacteria do move in response to different chemical gradients, so called chemotaxis. By this ability the cell can choose whether it wants tu "run" or "tumble", which are the 2 modes of bacterial propulsion known. When running the bacteria just go in a straight line. When the bacteria tumble they will randomly reorientate themselves, until the mode is switched back to run and they run off into another direction. The cells use these mechanisms so that they will increase the rate of tumbling in an unfavorable environment, so that they tumble a lot and thereby get away from the unfavorable environment. In a favorable environment the bacteria will reduce their tumbling frequency so as they won't leave the good environment they are in at the moment.
+
Bacteria's main form of propulsion in liquids is thorugh swimming by the use of flagella. A single flagellum is a thin filament around 100-150 Å thick, that extends many cell lengths out from the cell [https://parts.igem.org/Part:BBa_K343003#References [2]].
 +
The flagella are anchored to the cell body by a large, wheel-like protein complex spanning both inner and outer membrane [https://parts.igem.org/Part:BBa_K343003#References [4]]. Through this complex, subunits are secreted to the tip of the flagellar tube[https://parts.igem.org/Part:BBa_K343003#References [3]], thus elongating the filament. The membrane anchor also functions as a rotary engine, driven by the proton motive force [https://parts.igem.org/Part:BBa_K343003#References [4]], which can either rotate clockwise or counterclockwise.
 +
Spinning in the counterclockwise direction, the flagella will twist into a bundle in the shape of a corkscrew, and create a linear driving force [https://parts.igem.org/Part:BBa_K343003#References [4]], propelling the cell in a straight line through the liquid. This form of movement is termed run. Spun in the clockwise direction one might expect the cell to reverse, but this is not the case. Instead the flagellar bundle will unwind, thereby creating chaotic movement [https://parts.igem.org/Part:BBa_K343003#References [4]]. This movement reorients the cell randomly and is termed tumbling.  
  
The bacterial propulsion works with the help of flagellae, small bacterial motors that rotate. The 6 - 8 flagella of E. Coli bacteria will rotate counter clockwise when being in "run-mode" and three of them will bundle up to create one big flagellum. This results in a smooth-straight line pattern of movement. When the bacteria tumble the flagellae rotate clockwise, which breaks the bigger bundle up and makes the flagella whip around randomly instead of their normal rotation.
+
Different taxis pathways that steer cells towards favorable conditions and away from danger work by regulating the frequency of tumbling events [https://parts.igem.org/Part:BBa_K343003#References [5]], increasing tumbling in unfavorable environments and decreasing the tumbling rate under favorable conditions. This form of movement, combining tumbling and run, with regulation of the tumbling frequency is termed a biased random walk [https://parts.igem.org/Part:BBa_K343003#References [5]].
 +
 
 +
At the molecular level, increased tumbling is achieved through a phosphorylation cascade beginning with the binding of a repellant to a transmembrane receptor [https://parts.igem.org/Part:BBa_K343003#References [4]]. The receptor is linked to two proteins, CheW and CheA [https://parts.igem.org/Part:BBa_K343003#References [4]]. CheA is a histidine-kinase that will autophosphorylate when the repellant binds [https://parts.igem.org/Part:BBa_K343003#References [4]]. The phosphoryl group is then transferred to CheY[https://parts.igem.org/Part:BBa_K343003#References [4]]. The flagellar motor complex has high affinity for phosphorylated CheY (CheY-p), and binding reverses the mode of movement from run to tumble.  
 +
<br>
  
 
====Molecular mechanism of the photosensor====  
 
====Molecular mechanism of the photosensor====  
[[Image:Team-SDU-Denmark-SRII.png|250px|thumb|right|The fusion,chimera-protein coupled to the chemotaxis pathway. Figure taken from Trivedi et al.(2)]]
+
[[Image:Team-SDU-Denmark-SRII.png|250px|thumb|right|The fusion chimera-protein coupled to the chemotaxis pathway. Figure taken from Trivedi ''et al.''[2]]]
The photosensor acts directly on the tumbling frequency by effecting E. Coli's normal chemotaxis pathway.  
+
As described above, modification of taxis happens through modification of the bacterial tumbling frequency. The photosensor acts directly on the ''E. coli'''s chemotaxis pathway, which we outlined in the paragraph above.
When exposed to bluelight, the sensory rhodopsin II will absorb the photons and undergo a change in ultrastructure, which is being transduced through HtrII on to the Tar domain. This effects decreases the autophosphorylation of the CheA protein, which in turn again decreases the amount of phosphorylated CheY, which just means that less of it will get phosphorylated. If there is less of the CheY-P, then there is a smaller chance of one of these molecules binding to the flagellar motor and making it turn clockwise, thereby inducing a lowered tumbling frequency in the system. The photosensor can also act in the opposite way, inducing a higher tumbling rate in the bacteria. Which effect the sensor will have depends on where NpHtrII and StTar are fused in the HAMP domain. If the fusion contains 20 more basepair of the HtrII domain and 20 less of the Tar domain, the photosensor would have the opposite effect and would be increasing the autophosphorylation of CheA.
+
The Sensory Rhodopsin II, is fused to the N-terminal portion of the transducer protein HtrII. This in turn is fused to the cytoplasmic portion of the ''Salmonella'' chemotaxis receptor Tar [https://parts.igem.org/Part:BBa_K343003#References [2]], where the HAMP domains of the two proteins are located, to achieve a uniform response in the complex. It is the Tar protein that binds to CheW and CheA, which are part of ''E. coli'''s normal chemotaxis pathway.
 
+
When exposed to blue light, the sensory rhodopsin II will absorb the photons and therefore tilt one of its transmembrane helices outwards [https://parts.igem.org/Part:BBa_K343003#References [7]], which changes the ultrastructure of the linked HtrII protein, which also passes the signaling on to Tar. This leads to a lower rate of autophosphorylation of CheA, which in turn again decreases the amount of phosphorylated CheY. The extend of this response is dependent on the amount of methylated CheA. This means that the amount of phosphorylated CheY will be smaller than normally in ''E. coli''. If there is less of the CheY-P, then there is a smaller chance of one of these molecules binding to the flagellar motor and making it turn clockwise, thereby inducing a lowered tumbling frequency in the system. The photosensor can also act in the opposite way, inducing a higher tumbling rate in the bacteria. How the sensory rhodopsin II acts on the bacteria depends on where NpHtrII and StTar are fused in the HAMP domain. If the fusion contains 20 more basepairs of the HtrII domain and 20 less of the Tar domain, the photosensor would have the opposite effect and would be increasing the autophosphorylation of CheA [1]
===Usage and parameters===
+
<br>
The part requires retinal to work in E.Coli. This can be achieved through adding retinal to the liquid growth medium and/or the plates. Currently we are doing experiments on wether the part also functions with an internal retinal source, ie retinal synthesis in E. Coli.
+
On top of adding retinal the cells will have to be grown in the dark for at least two hours after the addition of retinal to the growth medium if you want to see an effect. This is so that the photosensor is not exposed to light before the experiments and will result in maximum output if exposed to blue light.
+
  
 +
==Usage and parameters==
 +
For information on the usage of the part and response in bacteria, please see composite part [[https://parts.igem.org/Part:BBa_K343007 K343007]], which is K343003 expressed under a constitutive promoter.<br><br>
  
 +
====Acknowledgements====
 +
Many thanks to Prof. Kwang-Hwang "Kevin" Jung for providing the physical DNA for this biobrick and the help with the part.<br><br>
 
<!-- -->
 
<!-- -->
<span class='h3bb'>Sequence and Features</span>
+
<span class='h3bb'>'''Sequence and Features'''</span>
 
<partinfo>BBa_K343003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K343003 SequenceAndFeatures</partinfo>
  
 +
===References===
 +
[1] Jung K-H, Spudich EN, Trivedi VD, Spudich JL, [http://jb.asm.org/cgi/content/short/183/21/6365 An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli], Journal of Bacteriology, November 2001, p. 6365-6371, Vol. 183, No. 21<br>
 +
[2] Samatey FA, et. al.,[http://www.nature.com/nature/journal/v410/n6826/abs/410331a0.html  Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling]Nature 410, 331-337 (15 March 2001)<br>
 +
[3] Berg HC, [http://www.annualreviews.org/eprint/cDJrS190m62mDRwHrlp9/full/10.1146/annurev.biochem.72.121801.161737 The rotary motor of bacterial flagella] Annual Review of Biochemistry Vol. 72: 19-54 (July 2003)<br>
 +
[4] Berg HC, [http://www.ncbi.nlm.nih.gov/pubmed/1098551 Chemotaxis in bacteria] Annu Rev Biophys Bioeng. 1975;4(00):119-36.<br>
 +
[5] Hess JF, Oosawa K, Kaplan N, Simon MI, [http://www.ncbi.nlm.nih.gov/pubmed/3280143 Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis.], Cell. 1988 Apr 8;53(1):79-87.<br>
 +
[6] Trivedi VD, Spudich JL, [http://www.ncbi.nlm.nih.gov/pubmed/14636056 Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phosphotransfer in vitro.], Biochemistry. 2003 Dec 2;42(47):13887-92.<br>
 +
[7] Subramanian S, Henderson R. [http://www.ncbi.nlm.nih.gov/pubmed/10949309 Molecular mechanism of vectorial proton translocation by bacteriorhodopsin] (2000) Nature 406, 653-657.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 23:05, 27 October 2010

NpSopII-NpHtrII-StTar (M-fusion)


Sensory Rhodopsin II bluelight receptor fused to its transducer, HtrII, with a 27 BP linker region. This is fused to Salmonella enterica serovar typhimurium chemotaxis protein Tar, so that the protein effectively couples the input from the receptor to the chemotaxis pathway and reduces the amount of phosphorylated CheY, which results in a lowered tumbling frequency. The fusion between HtrII and Tsr is an M-fusion in the HAMP domain, which is supposed to give it maximum activity. M-fusion emans that the last residue in the HtrII part of the fusion in the HAMP domain, consists of the aminoacid methionine. The other fusions which have been constructed are P- and G-fusions, named after the same principle. [1] Sequencing confirmed that the sequence is identical to that found in the article by Jung, Spudich E, Trivedi and Spudich J in the article: An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli. [1]

Background

The mechanism of bacterial motility

Bacteria's main form of propulsion in liquids is thorugh swimming by the use of flagella. A single flagellum is a thin filament around 100-150 Å thick, that extends many cell lengths out from the cell [2]. The flagella are anchored to the cell body by a large, wheel-like protein complex spanning both inner and outer membrane [4]. Through this complex, subunits are secreted to the tip of the flagellar tube[3], thus elongating the filament. The membrane anchor also functions as a rotary engine, driven by the proton motive force [4], which can either rotate clockwise or counterclockwise. Spinning in the counterclockwise direction, the flagella will twist into a bundle in the shape of a corkscrew, and create a linear driving force [4], propelling the cell in a straight line through the liquid. This form of movement is termed run. Spun in the clockwise direction one might expect the cell to reverse, but this is not the case. Instead the flagellar bundle will unwind, thereby creating chaotic movement [4]. This movement reorients the cell randomly and is termed tumbling.

Different taxis pathways that steer cells towards favorable conditions and away from danger work by regulating the frequency of tumbling events [5], increasing tumbling in unfavorable environments and decreasing the tumbling rate under favorable conditions. This form of movement, combining tumbling and run, with regulation of the tumbling frequency is termed a biased random walk [5].

At the molecular level, increased tumbling is achieved through a phosphorylation cascade beginning with the binding of a repellant to a transmembrane receptor [4]. The receptor is linked to two proteins, CheW and CheA [4]. CheA is a histidine-kinase that will autophosphorylate when the repellant binds [4]. The phosphoryl group is then transferred to CheY[4]. The flagellar motor complex has high affinity for phosphorylated CheY (CheY-p), and binding reverses the mode of movement from run to tumble.

Molecular mechanism of the photosensor

The fusion chimera-protein coupled to the chemotaxis pathway. Figure taken from Trivedi et al.[2]

As described above, modification of taxis happens through modification of the bacterial tumbling frequency. The photosensor acts directly on the E. coli's chemotaxis pathway, which we outlined in the paragraph above. The Sensory Rhodopsin II, is fused to the N-terminal portion of the transducer protein HtrII. This in turn is fused to the cytoplasmic portion of the Salmonella chemotaxis receptor Tar [2], where the HAMP domains of the two proteins are located, to achieve a uniform response in the complex. It is the Tar protein that binds to CheW and CheA, which are part of E. coli's normal chemotaxis pathway. When exposed to blue light, the sensory rhodopsin II will absorb the photons and therefore tilt one of its transmembrane helices outwards [7], which changes the ultrastructure of the linked HtrII protein, which also passes the signaling on to Tar. This leads to a lower rate of autophosphorylation of CheA, which in turn again decreases the amount of phosphorylated CheY. The extend of this response is dependent on the amount of methylated CheA. This means that the amount of phosphorylated CheY will be smaller than normally in E. coli. If there is less of the CheY-P, then there is a smaller chance of one of these molecules binding to the flagellar motor and making it turn clockwise, thereby inducing a lowered tumbling frequency in the system. The photosensor can also act in the opposite way, inducing a higher tumbling rate in the bacteria. How the sensory rhodopsin II acts on the bacteria depends on where NpHtrII and StTar are fused in the HAMP domain. If the fusion contains 20 more basepairs of the HtrII domain and 20 less of the Tar domain, the photosensor would have the opposite effect and would be increasing the autophosphorylation of CheA [1]

Usage and parameters

For information on the usage of the part and response in bacteria, please see composite part [K343007], which is K343003 expressed under a constitutive promoter.

Acknowledgements

Many thanks to Prof. Kwang-Hwang "Kevin" Jung for providing the physical DNA for this biobrick and the help with the part.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1783
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 73
    Illegal NgoMIV site found at 331
    Illegal AgeI site found at 1585
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1027
    Illegal BsaI.rc site found at 1300
    Illegal SapI site found at 801
    Illegal SapI.rc site found at 1801

References

[1] Jung K-H, Spudich EN, Trivedi VD, Spudich JL, [http://jb.asm.org/cgi/content/short/183/21/6365 An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli], Journal of Bacteriology, November 2001, p. 6365-6371, Vol. 183, No. 21
[2] Samatey FA, et. al.,[http://www.nature.com/nature/journal/v410/n6826/abs/410331a0.html Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling]Nature 410, 331-337 (15 March 2001)
[3] Berg HC, [http://www.annualreviews.org/eprint/cDJrS190m62mDRwHrlp9/full/10.1146/annurev.biochem.72.121801.161737 The rotary motor of bacterial flagella] Annual Review of Biochemistry Vol. 72: 19-54 (July 2003)
[4] Berg HC, [http://www.ncbi.nlm.nih.gov/pubmed/1098551 Chemotaxis in bacteria] Annu Rev Biophys Bioeng. 1975;4(00):119-36.
[5] Hess JF, Oosawa K, Kaplan N, Simon MI, [http://www.ncbi.nlm.nih.gov/pubmed/3280143 Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis.], Cell. 1988 Apr 8;53(1):79-87.
[6] Trivedi VD, Spudich JL, [http://www.ncbi.nlm.nih.gov/pubmed/14636056 Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phosphotransfer in vitro.], Biochemistry. 2003 Dec 2;42(47):13887-92.
[7] Subramanian S, Henderson R. [http://www.ncbi.nlm.nih.gov/pubmed/10949309 Molecular mechanism of vectorial proton translocation by bacteriorhodopsin] (2000) Nature 406, 653-657.