From 2010.igem.org

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