Part:BBa_K2835000

Enigneered mutant of mRFP1 + RBS (compatible with all iGEM RFC assembly standards)

This composite part is an improved version of BBa_I13502 (by MIT 2005) made compatible with assembly standard RFC25. Thereby, this part is compatible with all iGEM assembly standards. This improvement was made by iGEM18_Stockholm.

Usage and Biology

Fluorescent proteins are easily identified and measured and therefore has a wide range of applications. They are a group of proteins that are frequently used as reporters of expression. RFP is frequently used to monitor physiological processes, visualize protein localization, and detecting transgenic expression in vivo. In our project, we used RFP to indicate the success of a transfection or other procedures meant to introduce foreign DNA into a cell. The red fluorescent protein we have worked with emits light at wavelength 584 and exhibits a bright red color when expressed in E.coli (TOP10). There are many different variants of Fluorescent proteins, those most commonly used are GFP (Green fluorescent protein), RFP (Red fluorescent protein) and YFP (Yellow fluorescent protein).

Robert E. Campbell started with Discosoma RFP (DsRed) and evolved a faster folding, monomeric variant. See paper listed in source on page BBa_E1010. Codon optimized for expression in bacteria.

Characterization

Figure 1: Gel showing products from colony PCR. Well 1: Ladder. Well 2: Non-mutated Biobrick, BBa_I13502. Well 3: SAMURAI product 1. Well 4: SAMURAI product 2. Well 5: SAMURAI product 3, colony a. Well 6: SAMURAI product 3, colony b.

After SAMURAI, a site-directed mutagenisis technique where two point mutations were introduced to remove the two AgeI sites in the original BioBrick (BBa_I13502), the BioBrick products were ligated into pSB1C3 and transformed into Top10 E.coli. The obtained colonies were checked by colony PCR, to see if they contained the disered insert. This showed an insert of the correct size, around 800 bp, in two out of four colonies (see Figure 1).

The sequencing result of the SAMURAI products showed that both mutations had been introduced in SAMURAI product 1 (only one of the two mutations was found in SAMURAI product 2). Hence, both AgeI sites found in BBa_I13502 had been removed and this part was now compatible to assembly standard RFC25.

In order to show that our inserted mutations do not affect the functionallity of the protein mRFP1, SAMURAI product 1 (RBS + mutant of mRFP1) was assembled with a strong constitutitive promoter (BBa_J23119). This complex was 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 the figures below, where cultures with the original BioBrick (BBa_I13502, consisiting of BBa_E1010 and BBa_B0034) 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 is an improved version of the original part. It has equal fluorescent properties and is compatible with assembly standard RFC25.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]