From 2010.igem.org

Revision as of 11:42, 19 October 2010

Archeal Light Receptor

Parallely to the light-sensitive Pif3/PhyB-system, another implementation strategy caught our attention: The generation of our E. lemming by the fusion of an archean photoreceptor to a bacterial chemotactic transducer. This was successfully demonstrated by Jung et al. in 2001 [1], who fused the Natronobacterium pharaonis NpSRII (Np seven-transmembrane retinylidene photoreceptor sensory rhodopsins II) and their cognate transducer HtrII to the cytoplasmic domain of the chemotaxis transducer EcTsr of Escherichia coli.

Rhodopsins are photoreactive, membrane-embedded proteins, which are found not only in archaea, but in eubacteria and microbes as well. In Natronobacterium pharaonis, the NpSRII contains a domain of seven membrane-spanning helices, which carry out two distinct functions: Firstly, they serve as photo-inducible ion-pumps and secondly, as actors in the chemotaxis signaling network. This was proposed by Spudich et al. in 2001.

Overview on the fusion modules Archaeal modules are combined with an eubacterial light-sensitive module to generate a fully functional synthetic module.

Encouraged by this work, we cloned the archeal photoreceptor NpSRII as biobrick into the standardized vector pSB1C3 [http://partsregistry.org/Part:BBa_K220001 BBa_K22001]. For the expression (and experimental purposes), we used pACT3 (BamHI/HindIII) as plasmid backbone, which has a CmR gene (Chloramphenicol resistance) and is IPTG-inducibile (Isopropyl-ß-D-thiogalactopyranosid).

Primers for pACT3

Name: AR-BamHI-forward Scale: 0.04 umol Purification: Desalted 5' Mod.: None Inner Mod.: None 3' Mod.: None Type: DNA Sequence (5'-3'): GTG GAT CCA AGG AGA TAT ACA TAT GGT TGG TCT GAC CAC CCT G Length: 43 Aliquots: NONE

Name: AR-Hind-reverse Scale: Genomics Purification: Desalted 5' Mod.: None Inner Mod.: None 3' Mod.: None Type: DNA Sequence (5'-3'): GCA AGC TTT TAA CCG CTA TAA ATT G Length: 25 Aliquots: NONE

Sequence

LOCUS 1046531_aercheal_taxisphusio 1964 bp DNA

FEATURES Location/Qualifiers

    CDS             27..1937

                    /label="aercheal_taxisphusio"

ORIGIN

GAGCTCGAATTCGCGGCCGCTTCTAGATGGTTGGTCTGACCACCCTGTTTTGGCTGGGTG

CAATTGGTATGCTGGTTGGCACCCTGGCATTTGCATGGGCAGGTCGTGATGCAGGTAGCG

GTGAACGTCGTTATTATGTTACCCTGGTTGGTATTAGCGGTATTGCAGCAGTTGCATATG

CAGTTATGGCACTGGGTGTTGGTTGGGTTCCGGTTGCAGAACGTACCGTTTTTGTTCCGC

GTATTGATTGGATTCTGACAACTCCGCTGATTGTGTACTTCCTGGGTCTGCTGGCAGGTC

TGGATAGCCGTGAATTTGGTATTGTTATTACCCTGAATACCGTTGTTATGCTGGCAGGTT

TTGCCGGTGCAATGGTTCCGGGTATTGAACGTTATGCACTGTTTGGTATGGGTGCAGTTG

CATTTATTGGCCTGGTTTATTATCTGGTTGGTCCGATGACCGAAAGCGCAAGCCAGCGTA

GCAGCGGTATTAAAAGCCTGTATGTTCGTCTGCGTAATCTGACCGTTGTTCTGTGGGCAA

TTTATCCGTTTATTTGGCTGCTGGGTCCGCCTGGTGTTGCACTGCTGACCCCGACCGTTG

ATGTTGCACTGATTGTTTATCTGGATCTGGTTACCAAAGTGGGCTTTGGTTTTATTGCAC

TGGATGCAGCAGCGACCCTGCGTGCAGAACATGGTGCAAGCGCAAGCAATGGTGCCAGCG

CCAGCCTGAATGTTAGCCGTCTGCTGCTGCCTGGTCGTGTTCGTCATAGCTATACCGGTA

AAATGGGTGCAACCTTTGCATTTGTTGGTGCACTGACCGTTCTGTTTGGTGCAATTGCAT

ATGGTGAAGTTACCGCAGCAGCAGCCACCGGTGATGCAGCAGCCGTTCAAGAAGCAGCAG

TTAGCGCAATTCTGGGTCTGATTATTCTGCTGGGTATTAATCTGGGTCTGGTTGCAGCCA

CCCTGGGTGGTGATACCGCAGCAAGCCTGAGCACCCTGGCAGCAAAAGCAAGCCGTATGG

GTGATGGTGATCTGGATGTTGAACTGGAAACCCGTCGTGAAGATGAAATTGGTGATCTGT

ATGCAGCCTTTGATGAACTGAAACGCATTAAAATTGTGACCAGCCTGCTGCTGGTTCTGG

CAGTTTTTGGTCTGCTGCAACTGACCAGCGGTGGTCTGTTTTTTAATGCACTGAAAAACG

ATAAAGAAAATTTTACCGTGCTGCAAACCATTCGCCAGCAGCAGAGCACCCTGAATGGTA

GCTGGGTTGCCCTGCTGCAAACCCGTAATACCCTGAATCGTGCAGGTATTCGTTATATGA

TGGACCAGAATAATATTGGTAGCGGTAGCACCGTTGCAGAACTGATGGAAAGCGCCAGCA

TTAGCCTGAAACAGGCAGAAAAAAACTGGGCAGATTATGAAGCACTGCCTCGTGATCCGC

GTCAGAGCACCGCAGCAGCCGCAGAAATTAAACGTAACTATGATATCTACCATAACGCAC

TGGCAGAACTGATTCAGCTGCTGGGTGCAGGTAAAATCAACGAATTTTTTGATCAGCCGA

CCCAGGGTTATCAGGATGGTTTTGAAAAACAGTATGTGGCCTACATGGAACAGAATGATC

GTCTGCATGATATTGCCGTGAGCGATAATAATGCAAGCTATAGCCAGGCAATGTGGATTC

TGGTTGGTGTTATGATTGTTGTTCTGGCCGTTATTTTTGCCGTGTGGTTTGGTATTAAAG

CAAGCCTGGTTGCACCGATGAATCGTCTGATTGATAGCATTCGTCATATTGCCGGTGGTG

ATCTGGTTAAACCGATTGAAGTTGATGGCAGCAATGAAATGGGTCAGCTGGCAGAAAGCC

TGCGTCATATGCAGGGTGAACTGATGCGTACCGTTGGTGATGTTCGTAATGGTGCAAATG

CAATTTATAGCGGTTAATACTAGTAGCGGCCGCTGCAGGGTACC

//

Chrystal structure of NpSRII

Sensory Rhodopsin II photocycle

References

[1] Jung, Spudich, Trivedi and Spudich: An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli. Journal of bacteriology. 2001; 21.

[2] Hartmut Luecke1 and Brigitte Schobert, Janos K. Lanyi, Elena N. Spudich, John L. Spudich. Crystal Structure of Sensory Rhodopsin II at 2.4 Angstroms: Insights into Color Tuning and Transducer Interaction. Science Express. 2001; 293;5534

[3] Shimono K, Iwamoto M, Sumi M, Kamo N.: Functional expression of pharaonis photorhodopsin in Escherichia coli. FEBS Lett 1997; 22

[4] Michael Hein, Ansgar A. Wegener,† Martin Engelhard,† and Friedrich Siebert. Time-Resolved FTIR Studies of Sensory Rhodopsin II (NpSRII) from Natronobacterium pharaonis: Implications for Proton Transport and Receptor Activation. Biophys J. 2003; 84

[5]Igor Chizhov, Georg Schmies, Ralf Seidel, Jens R. Sydor, Beate Lüttenberg and Martin Engelhard.The Photophobic Receptor from Natronobacterium pharaonis: Temperature and pH Dependencies of the Photocycle of Sensory Rhodopsin II. Biophysical Journal. 1898; 75(2)

[6] Jones, P. C., and R. H. Fillingame. 1998. Genetic fusions of subunit c in the F0 sector of H+-transporting ATP synthase. Functional dimers and trimers and determination of stoichiometry by cross-linking analysis. J. Biol. Chem. 273:29701-29705