Difference between revisions of "Part:BBa K786001:Design"

Latest revision as of 17:37, 28 September 2012

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

Design and Construction Notes

Design strategy

Pfam (version 26.0) was used to predict the domain of fusion protein. Linker was refereed and modified from previous research study of SRII fusion with HtrII.

Restriction sites of HindIII and BamHI were added before and after the SRII gene respectively for two reasons. 1. Enable further integration of other peptides (e.g.: His-tag), for the construction of a larger fusion protein. (HindIII for N-terminal while, BamHI for C-terminal) 2. Enable for switching the sensory rhodopsin portion of the fusion protein. According to previous study. [2] A series of mutant sensory rhodopsins have been identified which covers a large variation of absorbing spectrum. 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.

The speI RE site after the promoter was kept so that the constitutive promoter can be switched to strictly controlled promoters such as PTET, tetracycline-inducible promoter; PBAD, arabinose-inducible promoter.

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

1. TGAAAGAGGAGAAATACTAGAAGCTTATGGTGGGACTTACGACCCT
2. CGCCGACGCGCCGTTCGACGCGGATCCGTCGGCGACCGCAGGCGTGT
3. GGATCCGCGTCGAACGGCGCGTCGGCGATGTCGCTGAACGTATCACG
4. TGCGCCAGTCGGTGCGGACAACCGTCGGTGATGTGCGCAA
5. CTACACTAGCACTATCAGCGTTAAAATGTTTCCCAGTTCT
6. AGAACTGGGAAACATTTTAACGCTGATAGTGCTAGTGTAG
7. AGGGTCGTAAGTCCCACCATAAGCTTCTAGTATTTCTCCTCTTTCA
8. TCGCGGACATGAGTGACGGTTGTCCGCACCGACTGGCGCA
9. TGCGCCAGTCGGTGCGGACAACCGTCACTCATGTCCGCGA
10. CTACACTAGCACTATCAGCGTCAAAATGTTTCCCAGTTTG
11. CAAACTGGGAAACATTTTGACGCTGATAGTGCTAGTGTAG
12. TGAAAGAGGAGAAATACTAGAAGCTTATGGACGCCGTCGCAACCGC
13. TGCGCCAGTCGCTTCGTGGCACCGTCACTCATGTCCGCGA
14. ATTCGCGGCCGCTTCTAGAGTCCCTTGCATTTACATTTTG
15. ATCTAGTATTTCTCCTCTTTAGTCCATTCTCCCCAAAAAT
16. CTAAAGAGGAGAAATACTAGATGGCTTCCTCCGAAGACGT
17. CAAAATGTAAATGCAAGGGACTCTAGAAGCGGCCGCGAAT
18. GGAAAGAGGAGAAATACTAGATGGCCACCACCGTACAACT
19. CTAATGATGATGATGATGATGCCCTTCTTTTGTCATGCCCT
20. CATCATCATCATCATCATTAGTACTAGTAGCGGCCGCTGCA
21. ATCTAGTATTTCTCCTCTTTCCGGACCGCAGGCTGGCTAG

Negative Phototactic Construct for Blue Light Detection
BBa_K786001

Primers 1 and 2 were used to amplify sensory rhodopsin II coding sequence from the genome of Natronomonas Pharaonis DSM 2160. Restriction sites of HindIII and BamHI were added.

Restriction sites of HindIII and BamHI were added before and after the SRII gene respectively in order to:

1. 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).
2. 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 4, 5 were used to amplify the coding sequence of Tsr from E. coli K-12genome.

Primers 6, 7 were used to amplify the promoter J23100 and J61002 backbone from biobrick BBa_J23100.

All of the parts amplified were added in PCR mix with equal molar to perform overlapping PCR and 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.

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] ung KH, Spudich EN, Trivedi VD, Spudich JL (2001). An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli. J Bacteriol. 183: 6365-6371 .