Genetic part – The BiFC Toolbox

GFP is the most popular fluorescent protein. In 2006 Pédelacq et al., engineered and characterize new form of GFP – superfolder GFP (sfGFP) – that is more stabile and glows stronger than wild GFP. We wanted to create another superfolder fluorescent proteins and we did it! Changing color of fluorescent proteins is possible by one-point mutagenesis. We did it with PCR. Our template was sfGFP from Parts Registry (BBa_I746908).

sfBFP (K1093000) - sfYFP (K1093001) - contains T203Y point mutation compared to the original sfGFP sfCFP (K1093002) -

Doing this we improved sfGFP (by giving it another color). Also we can say that we improved standard forms of CFP, BFP and YFP by creating their superfolder forms. We send sfBFP, sfYFP and sfCFP to Parts Registry.

When we confirmed our constructs by sequencing, we put it at on pSB1A3 plasmid, added J23100 promoter and B0034 RBS and measure them in RF. We chosen RF, because our standard medium, LB, shined to much to visualize sfBFP in liquid.

Excitation/emission peak for each protein: - sfGFP – 485/510 nm - sfBFP – 380/445 nm - sfCFP – 425/475 nm - sfYFP – 503/540 nm

Pic 1. – Glowing of sfCFP, sfYFP and sfBFP compared to sfGFP (BBa_I746908).

We compared glowing to shining level of sfGFP (in our measure it is 100% glowing).

	samples	arithmetic mean	standard deviation

sfGFP 100,60 100,37 99,02 100,00 0,85 sfCFP 17,95 18,38 17,36 17,90 0,51 sfBFP 40,65 41,52 42,25 41,47 0,80 sfYFP 218,87 216,51 217,87 217,75 1,19 Tab 1. – Glowing of sfCFP, sfYFP and sfBFP compared to sfGFP (BBa_I746908).

We found in Parts Registry yellow fluorescent protein, named SYFP2 (super yellow fluorescent protein 2; BBa_ K864100). We decided to compare it with our sfYFP. We put BBa_K864100 on pSB1A3 plasmid, add J23100 promoter and B0034 RBS, and measure it in RF. Excitation/emission peak: 503/540 nm

Pic 2. – Glowing of sfYFP and SYFP2 compared to sfGFP (BBa_I746908).

	samples	arithmetic mean	standard deviation

sfGFP 100,60 100,37 99,02 100,00 0,85 sfYFP 218,87 216,51 217,87 217,75 1,19 SYFP2 424,70 418,26 412,40 418,45 6,15 Tab 2. – Glowing of sfYFP and SYFP2 compared to sfGFP (BBa_I746908).

Honestly, we aren’t happy that our sfYFP isn’t glowing as strong as SYFP2. But we sincerely congratulate iGEM2012 Uppsala Team for creating great yellow fluorescent protein.

We created superfolder BFP, CFP and GFP because, we wanted to make The BiFC Toolbox. BiFC (Bimolecular Fluorescent Complementation) is method used to validate and visualize protein-protein interactions in living cells. Fragments of fluorescent proteins are fused to proteins that we study and if they interact, fluorescent protein folds, reform in its native structure and emit signal. Superfolder proteins are perfect for BiFC system, because they are stabile and fold very quickly.

We cut sfGFP, sfCFP, sfBFP and sfYFP by PCR using specific primers. N-terminal fragment is long (645 bp) and specific to each protein. C-terminal fragment is very short (54 bp) and comes from sfGFP, but we suspect that it would be working with other fluorescent proteins. C-terminal is in two variants: m6 and m12. Also we prepared to BiFC system wild GFP (E0040), mCherry (J06504) and mOrange (E2050).

We successful amplificated N-sfBFP, N-sfYFP, N-sfCFP, N-mCherry, C-mCherry and cloned them to pSB1C3 plasmid. We send them to Parts Registry.

To verification our BiFC fragments, we wanted to use b-Fos and b-Jun, that interacts with themselves. We wanted to fuse b-Jun with N-terminal fragment and b-Fos with C-terminal. Also, we wanted to use b-Fos without leucine zipper (without it, it isn’t interact with b-Jun) as negative control. To measurements we’ll lize bacteria, that express b-Fos or b-Jun fused with part of fluorescent protein and mix it.

Unfortunately, vacation is over and we couldn’t fulfill our dreams and measure our constructs.

Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS, 2006, Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology 24(1):79-88. Shyu YJ, Liu H, Deng X, Hu C, 2006, Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions. BioTechniques 40:61-66.