Part:BBa_K2276009

pTetR-mOrange

Biobrick BBa_K2276009 is a device, derived from BBa_E2050. Based on the part BBa_E2050 which containing coding sequence of mOrange only, we added pTetR promotor(BBa_R0040), RBS(BBa_B0034) and double terminators(BBa_B0010 and BBa_B0012) to it.

mOrange a fluorescent protein, derived from mRFP (DsRed). It has a higher quantum yield. However mOrrange is blue shifted and mature more slowly compared to current (1/05) best red, mCherry.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 843
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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, which contribute to the expression of mOrange in E. coli.

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.

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.

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).

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).

Fig.1 Expression of mOrange in E. coli BL21(DE3) strain. (a), E. coli BL21(DE3) cells are transformed with ① part BBa_K2276009, ② recombinant plasmid pCI-luxI-pSB1C3, or ③ nothing and then cultured in LB solid media containing chloramphenicol for about 18 hours. (b), E. coli BL21(DE3) cells are transformed with ① part BBa_K2276009 or ② recombinant plasmid pCI-luxI-pSB1C3 and then cultured in LB liquid media containing chloramphenicol for about 24 hours. ③, negative control, LB liquid media containing chloramphenicol

Source

BBa_E2050

References

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