Difference between revisions of "Part:BBa K786003:Design"

Revision as of 16:59, 28 September 2012

Sensory rhodopsin I (SRI) with HtrII & Tar, sensitive to orange light

Design and Construction Notes

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

Phototactic construct for orange light detection 

BBa_K786003

<img src="" width="606" height="130" style="margin:15px" />

       SRI was fused with HtrI with a linker peptide, where only the membrane-proximal cytoplasmic domain of the native HtrI was kept, while the cytoplasmic domains were replaced by that of Tar. Once the fusion protein was triggered, the histidine kinase (CheA) will be regulated, leading to phototactic effect. 

<img src="" width="335" height="436" style="margin:15px" />

Source

From genomic sequence of Halobacterium Salinarum and E.coli K12.

References