Part:BBa_K2835007

Constitutive reporter: strong promoter + RBS + mRFP1

This constitutive reporter consists of strong constitutive promoter (BBa_J23119) and a part contaning RBS + mRFP1 (BBa_I13502).

The part BBa_K2835001 (including a constitutitive promoter BBa_J23119) was composed for protein expression in order to show that the two inserted point mutations in our improved BioBrick BBa_K2835000 (RBS + mutant of mRFP1) would not negatively affect the functionallity of the protein mRFP1. For experimental comparison, the original BioBrick (BBa_I13502) was also assembled with a the promoter BBa_J23119, resulting in this BioBrick. These complexes were transformed into Top10 E.coli, resulting in red colonies. Liquid cultures of the inoculated red colonies showed a bright red color after one night incubation, as can bee seen in Figure 1, where cultures with the original BioBrick (BBa_I13502) and our improved version (BBa_K2835000) are shown side by side. To prove that the introduced mutations did not change the fluorescence of the protein, absorbance measurements were performed. A wavelength of 600 nm was used to check the cell density, while 584 nm (excitation peak of mRFP1) was used to measure the red fluorescent protein. The results showed close to identical absorbance for both versions of mRFP1, with values of OD₅₈₄/OD₆₀₀ = 1.058 for the original BioBrick and OD₅₈₄/OD₆₀₀ = 1.068 for our mutated version. These results, together with sequencing results confirming the introduced mutations, prove that our mutated BioBrick (BBa_K2835000) is an improved version of the original part (BBa_I13502). It has equal fluorescent properties and is compatible with assembly standard RFC25.

Figure 1: Liquid overnight cultures (Top10 E.coli) with the original BioBrick encoding mRFP1, BBa_I13502, and our improved version of this BioBrick, BBa_K2835000. Both parts have been assembled with a strong constitutive promoter (BBa_J23119).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 616
  Illegal AgeI site found at 728
  
• 1000
  COMPATIBLE WITH RFC[1000]