Genetic part – The BiFC Toolbox

GFP is the most popular fluorescent protein. In 2006 Pédelacq et al., engineered and characterized new form of GFP – superfolder GFP (sfGFP), which is more stabile and has stronger fluorescence than wild GFP. We aimed to create another superfolder fluorescent proteins and we did it! Changing color of fluorescent proteins is possible using directed mutagenesis technique. We made it using 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) -

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

When we confirmed the viability of our constructs by sequencing, we put it at on pSB1A3 plasmid, added J23100 promoter and B0034 RBS and subsequently measure them in RF. We chose RF, because our standard medium, LB, have high internal fluorescence, which make impossible to visualize sfBFP in liquid without high experimental error.

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

Pic 1. – Fluorescence 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. – Fluorescence 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. – Fluorescence of sfYFP and SYFP2 compared to sfGFP (BBa_I746908).

The fact that fluorescence of sfYFP is not as strong as fluorescence of SYFP2 is slighty disappointing. However, we sincerely congratulate iGEM2012 Uppsala Team for creating great yellow fluorescent protein.

We created superfolder BFP, CFP and GFP because, we aimed 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, functional fluorescent protein is formed and a signal fluorescent signal is emitted. Superfolder proteins are perfect for BiFC system due to their improved stability and relatively fast folding kinetics.

We cut sfGFP, sfCFP, sfBFP and sfYFP by PCR using specific primers. N-terminal fragment is long (645 bp) and specific to each protein. On the other hand C-terminal fragment is very short (54 bp) and comes from sfGFP, but we expect that it would be working with other fluorescent proteins. C-terminal is in two variants: m6 and m12. Moreover, 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 and we sent them to Parts Registry.

To verify the functionality of our BiFC fragments, we decided to use b-Fos and b-Jun, that interact with themselves. We aimed to fuse b-Jun with N-terminal fragment and b-Fos with C-terminal. Furthermore we intended to use b-Fos without leucine zipper (it such experimental condition it is not able to interact with b-Jun) as negative control. To measurements we lysed bacteria expressing b-Fos or b-Jun fused with part of fluorescent protein and mix both lysates.

Unfortunately, we had not enought time and we do not manage to fulfill our ultimate aim and measure our constructs.

References:

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.