Part:BBa_K2835001

Constitutive reporter: strong promoter + RBS + mutant of mRFP1 (RFC25 compatible)

This constitutive reporter consists of strong constitutive promoter BBa_J23119 and a translational unit BBa_K2835000 (RBS + mutant of mRFP1).

Usage and Biology

Fluorescent proteins are easily identified and measured and therefore have a wide range of applications. They are a group of proteins that are frequently used as reporters of expression. RFP is commonly used to monitor physiological processes, visualize protein localization, and detecting transgenic expression in vivo. In our project, we explored SAMURAI, a site-directed-mutagenisis technique, and used this method to introduce two point mutations in the BioBrick BBa_I13502 (RBS + mRFP1) to remove its two AgeI sites, therebey creating an imporved BioBrick, BBa_K2835000, compatible with assembly standard RFC25. The improved BioBrick (BBa_K2835000) was assembled with a strong constitutive promoter in order to express the mRFP1 protein, resulting in this BioBrick.

Characterization

This part was composed for protein expression in order to show that the two inserted point mutations in our improved BioBrick BBa_K2835000 (RBS + 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 same promoter (BBa_J23119), resulting in a BioBrick named BBa_K2835007. 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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

color	Red
emission	607 nm
excitation	584 nm