Part:BBa_E2050

derivative of mRFP1, yeast-optimized

mRFP derivative. Ex548nm/Em562. yeast codon optimized.

Usage and Biology

mOrange, a mRFP (DsRed) derivative. Higher quantum yield but blue shifted and slower maturing compared to current (1/05) best red, mCherry.

We used the part BBa_E2050to construct mRFP-fused protein. Sequencing result revealed this part is working.

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Tianjin IGEM Team

Sequence and Features

Barcodes are discontinued, but one was appended to the sequence of this part. Composite parts using this part will include the barcode. More ...

Assembly Compatibility:

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

  Illegal BglII site found at 763
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

The followings were edited by SCU_China-2017

Improvement

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.

Characterization

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