Difference between revisions of "Part:BBa K2835000"

Latest revision as of 22:35, 16 October 2018

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

This translational unit 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

Figure 1: mRFP1 expressed in Top10 E.coli

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 used mRFP1 to indicate the success of a transfection or other procedures meant to introduce foreign DNA into a cell. This red fluorescent protein (mRFP1) has an excitation peak at a wavelength of 584 nm and exhibits a bright red color when expressed in E.coli. 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 (mRFP1).

Characterization

Figure 2: 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.

SAMURAI, a site-directed mutagenisis technique, was used to introduce two point mutations in BBa_I13502 with the aim of removing its two AgeI sites. The products of SAMURAI, hopefully mutated and improved versions of BBa_I13502, were ligated into the plasmid pSB1C3 and transformed into Top10 E.coli. The obtained colonies were checked by colony PCR, to see if they contained the desired insert. This showed an insert of the correct size, just under 800 bp, in two out of four colonies (see Figure 2).

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 in SAMURAI product 1 and this part was now compatible to assembly standard RFC25.

In order to show that our inserted mutations did 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 Figure 3, 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 is an improved version of the original part. It has equal fluorescent properties and is compatible with assembly standard RFC25.

Figure 3: 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).

References

Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, et al. A monomeric red fluorescent protein. 2002;99(12).

Sequence and Features

Functional Parameters