Part:BBa_K786010:Design
Gene expression for sensory rhodopsin I & II (SRI & SRII)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 659
Illegal AgeI site found at 771 - 1000COMPATIBLE WITH RFC[1000]
Design and Construction Notes
Strong RBS was used to allow high expression of visible fluorescence protein once the sensory rhodopsin I & II (SRI & SRII) detect the correct light spectrum.
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
The Gene Expression System
BBa_K786010
Primers 14 and 15 were used to amplify the promoter R0083 from the biobrick BBa_R0083. Primers 16 and 17 were used to amplify the back bone pSB1C3 and the RFP reporter gene by using biobrick BBa_K606030. Strong RBS BBa_B0034 was added in the construct during the primer design.
Source
All are constructed from previous biobricks
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] Hoff WD, Jung KH, Spudich JL (1997). Molecular mechanism of photosignaling by archaeal sensory rhodopsins. Annu Rev Biophys Biomol Struct. 26: 223-258.
[5] Spudich JL, Yang CS, Jung KH, Spudich EN (2000). Retinylidene proteins: structures and functions from archaea to humans. Annu Rev Cell Dev Biol. 16: 365-392.
[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.