Team:ETHZ Basel/Biology/Archeal Light Receptor
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.
Encouraged by this work, we cloned the archeal photoreceptor NpSRII as biobrick into the standardized vector pSB1C3 BBa_K422001. 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 //