Part:BBa_K786010:Design

Gene expression for sensory rhodopsin I & II (SRI & SRII)

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 659
  Illegal AgeI site found at 771
  
• 1000
  COMPATIBLE 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 [1] 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

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][1] 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.