Part:BBa_K3287002
Cu_Blue
This composite part is a cooper biosensor. It is composed of the cueR activator under the control of a constitutive promoter, the cooper specific promoter copAP, a strong rbs, the amilCP blue chromoprotein and a transcription terminator. In presence of cooper, bacteria turn into different blue color intensities according to the concentration of cooper.
TITULO
Our biosensor for cooper detection is composed of a regulatory sequence made up by the CueR activator and the cooper specific promoter copAP. This promoter is regulated by CueR, which binds Cu2+ ions (Yamamoto and Ishihama 2005), that is under the control of a constitutive promoter (BBa_K608002). Then we used a blue chromoprotein (amilCP BBa_K592009) downstream the promoter for a first sight detection, with a strong RBS (BBa_B0030). The correct construction of this plasmid was confirmed by sequencing (Figure 1).
[[File:|600px|thumb|center|Figure 1. Figure 1. Construction of expression vector Cu_Blue (BBa_3287002) from parts of 2015_Bielefeld-CeBiTec: BBa_K1758320 and BBa_K1758323 (only the copAP promoter sequence); and amilCP coming from the chromoprotein collection of 2011_Uppsala-Sweden team. The CueR-copAP-amilCP composite part is cloned pSB1C3 vector through the BioBrick suffix-prefix site.]]
We transformed the plasmid containing BBa_K3287002 into E. coli competent cells and cultured at 37ºC until OD = 0.4. Then we add CuCl2 at different concentrations to induce the expression at 37 ℃ for 6 hours. After that, 2 mL of the bacterial culture were centrifuged at 3,000 r.p.m for 3 minutes, so we could observe at first sight the result of copAP promoter being activated by cooper (Figure 2).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 475
Illegal SapI.rc site found at 625
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