Part:BBa_K2276009

pTetR-mOrange

Biobrick BBa_K2276009 is a device, derived from BBa_E2050. It consists of pTetR promotor, RBS, terminator and the coding sequence of fluorescent protein mOrange. mOrange is derived from mRFP (DsRed). It has higher quantum yield. However mOrrange is blue shifted and mature more slowly compared to current (1/05) best red, mCherry.

Sequence and Features No part name specified with partinfo tag.

Biology

mOrange is a fluorescent protein derived from mRFP (DsRed) with higher quantum yield. It is blue shifted and mature more slowly compared to current (1/05) best red, mCherry. The original part BBa_E2050 just contains coding sequence of mOrange. However, the new part BBa_K2276009 contains not only coding sequence but also promoter, RBS and terminator. Therefore, mOrange can be expressed in E. coli.

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Results

In order to observe the fluorescence of mOrange easily and obviously, we transformed plasmid containing this part into E. coli BL21(DE3) strain which doesn’t have tetR gene. In this circumstance, mOrange can be expressed in BL21(DE3) cells constitutively.

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After transformation, BL21(DE3) cells were cultured in LB solid media containing chloramphenicol for about 18 hours. We used two controls in the whole process, BL21(DE3) competent cells transformed with recombinant plasmid pCI-luxI-pSB1C3 and BL21(DE3) competent cells transformed with nothing.

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BL21(DE3) colonies transformed with part BBa_K2276009 are orange. BL21(DE3) colonies transformed with pCI-luxI-pSB1C3 are in normal color. BL21(DE3) cells without any other plasmids can’t grow on plate containing chloramphenicol(Figure 1, a).

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And then, we isolated the single colonies from selective plates, and inoculated a culture of about 3 mL LB medium containing chloramphenicol. The cultures were incubated at 37℃ for about 24 hours. Culture of BL21(DE3) cells transformed with part BBa_K2276009 are orange obviously. And BL21(DE3) cells transformed with pCI-luxI-pSB1C3 are light yellow(Figure 1, b).

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Source

BBa_E2050

References

[1] Shaner et al, 2004. Nat Biotech (22):1567-1571