Part:BBa_K786002:Design

Sensory rhodopsin II (SRII) with HtrII & Tar, sensitive to blue-green light

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal SpeI site found at 37
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal SpeI site found at 37
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 785
23
INCOMPATIBLE WITH RFC[23]
Illegal SpeI site found at 37
25
INCOMPATIBLE WITH RFC[25]
Illegal SpeI site found at 37
Illegal NgoMIV site found at 140
Illegal NgoMIV site found at 398
Illegal AgeI site found at 1703
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1139
Illegal SapI site found at 913

Design and construction Notes

Biobrick Construction Strategy
The Sensory Rhodopsin system works in E.coli by fusing SR and Htr with a flexible linker and joint the HtrII membrane-proximal cytoplasmic fragment with the cytoplasmic domain (Methyl-accepting chemotaxis protein (MCP) signalling domain) of the eubacterial chemotaxis receptor.[1] The fusion protein in BBa_K317028 and BBa_K317003 used the Tar gene from Synechocystis typhimurium, have their construct design based on [2]. The previous study made three different junction constructs (P, G, M) based on studies of dimerization, yet no accurate tool was used for domain determination. Therefore, we made the following improvements.

1. Tar gene from E.coli K12 strain was used instead of that of S.typhimurium, as we believe E.coli can express and function more properly with its native genes and proteins.
2. We designed the construct by using an accurate protein domain determining tool [3], the whole E.coli Tar protein consist of three domains- Tar ligand binding domain (amino acid sequence 1-175), the HAMP domain (amino acid sequence 194-263) and the Methyl-accepting chemotaxis protein (MCP) signalling domain (amino acid sequence 264-553). As only the cytoplasmic domain is needed, the exact Methyl-accepting chemotaxis protein (MCP) signalling domain (amino acid sequence 264-553 of EcTar) was cut and fused with membrane-proximal cytoplasmic fragment part of HtrII (amino acid sequence 1- 133).
Method of construction
Our team made use of a fast and convenient assembly method developed recently [4] to construct all of our biobricks in an effective way without the use of restriction enzymes and ligase - the direct transformation of prolonged overlap extension PCR products.

Amplification of genes
Linear fragment DNA of the insert(s) and vector were amplified from corresponding templates by using specially designed primers which can add overlapping regions (40 bps per linear DNA) onto the DNA fragments.

Prolonged overlap extension PCR
Equal molar of insert(s) and vector DNA were added into a PCR reaction mix. The POE-PCR was conducted as follows: denaturation at 98°C for 30 s; 25 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 2.5 min.

Direct Transformation
Five microliter of the prolonged overlap extension PCR products was used to transform competent cells directly.

Constructs

List of primers

Primer# primer sequence

TGAAAGAGGAGAAATACTAGAAGCTTATGGTGGGACTTACGACCCT
CGCCGACGCGCCGTTCGACGCGGATCCGTCGGCGACCGCAGGCGTGT
GGATCCGCGTCGAACGGCGCGTCGGCGATGTCGCTGAACGTATCACG
TGCGCCAGTCGGTGCGGACAACCGTCGGTGATGTGCGCAA
CTACACTAGCACTATCAGCGTTAAAATGTTTCCCAGTTCT
AGAACTGGGAAACATTTTAACGCTGATAGTGCTAGTGTAG
AGGGTCGTAAGTCCCACCATAAGCTTCTAGTATTTCTCCTCTTTCA
TCGCGGACATGAGTGACGGTTGTCCGCACCGACTGGCGCA
TGCGCCAGTCGGTGCGGACAACCGTCACTCATGTCCGCGA
CTACACTAGCACTATCAGCGTCAAAATGTTTCCCAGTTTG
CAAACTGGGAAACATTTTGACGCTGATAGTGCTAGTGTAG
TGAAAGAGGAGAAATACTAGAAGCTTATGGACGCCGTCGCAACCGC
TGCGCCAGTCGCTTCGTGGCACCGTCACTCATGTCCGCGA
ATTCGCGGCCGCTTCTAGAGTCCCTTGCATTTACATTTTG
ATCTAGTATTTCTCCTCTTTAGTCCATTCTCCCCAAAAAT
CTAAAGAGGAGAAATACTAGATGGCTTCCTCCGAAGACGT
CAAAATGTAAATGCAAGGGACTCTAGAAGCGGCCGCGAAT
GGAAAGAGGAGAAATACTAGATGGCCACCACCGTACAACT
CTAATGATGATGATGATGATGCCCTTCTTTTGTCATGCCCT
CATCATCATCATCATCATTAGTACTAGTAGCGGCCGCTGCA
ATCTAGTATTTCTCCTCTTTCCGGACCGCAGGCTGGCTAG

Positive Phototactic Construct for Blue Light Detection
BBa_K786002

Primers 1 and 2 were used to amplify sensory rhodopsin II (SRII) coding sequence from the genomic DNA of Natronomonas Pharaonis DSM 2160. Restriction sites of HindIII and BamHI were added. [remark 1]
Restriction sites of HindIII and BamHI were added before and after the SRII gene respectively in order to:

Enable further integration of other peptides such as His-tag, or construct a larger fusion protein (HindIII for N-terminus ligation while BamHI for C-terminus).
Enable us to switch the sensory rhodopsin portion of the fusion protein. A series of mutant sensory rhodopsins were identified which cover a large variation of absorption spectrum [5]. These two restriction sites allow further switching of the sensing unit, so the light sensing system can be tuned for sensing different kinds of light source.

Primers 3, 8 were used to amplify the coding sequence of HtrII from the genome of Natronomonas Pharaonis DSM 2160. A linker (GSASNGASA) that was proven not affecting the SR system [6] was added to joint SRII and HtrII.

Primers 9, 10 were used to amplify the coding sequence of Tar from E. coli K-12 genomic DNA.

Primers 7, 11 were used to amplify the promoter J23100 and J61002 backbone from biobrick BBa_J23100.

All of the parts amplified were added into a single PCR mix with equal molar to perform overlapping PCR. The PCR product was used for direct transformation.

The insert was later on switched to pSB1C3 backbone by using EcoRI and PstI restriction enzymes and T4 ligase. [Remark 2]
The SpeI site after the promoter was kept so that the constitutive promoter can be switched to strictly controlled promoters such as Ptet (tetracycline-inducible promoter) and PBAD (arabinose-inducible promoter).

Source

From genomic sequence of bacterial N. pharaonis (DSM 2160) and E.coli K12.

References

[1] Vishwa D. Trivedi and John L. Spudich. Photostimulation of a Sensory Rhodopsin II/HtrII/Tsr Fusion Chimera Activates CheA-Autophosphorylation and CheY-Phosphotransfer in Vitro. Biochemistry 2003, 42, 13887-13892 BBa_K317028

[2] KWANG-HWAN JUNG, ELENA N. SPUDICH, VISHWA D. TRIVEDI, AND JOHN L. SPUDICH, An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli, JOURNAL OF BACTERIOLOGY, Nov. 2001, p. 6365–6371

[3] Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K, Holm L, Sonnhammer EL, Eddy SR, Bateman A., The Pfam protein families database., Nucleic Acids Res. 2010 Jan;38(Database issue):D211-22.

[4]You C, Zhang XZ, Zhang YH (2012). Simple cloning via direct transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis. Appl Environ Microbiol. 78: 1593-1595.

[5] Sudo Y, Yuasa Y, Shibata J, Suzuki D, Homma M (2011). Spectral tuning in sensory rhodopsin I from Salinibacter ruber. J Biol Chem. 286: 11328-11336.

[6] Welch M, Oosawa K, Aizawa S, Eisenbach M (1993). Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria. Proc Natl Acad Sci U S A. 90: 8787-8791.