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 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 direcred mutagenesis technique. We made it using PCR. Our template was sfGFP from Parts Registry (BBa_I746908).

• sfBFP (K1093000) - contains Y66H point mutation compared to the original sfGFP
• sfYFP (K1093001) - contains T203Y point mutation compared to the original sfGFP
• sfCFP (K1093002) - contains Y66W point mutation compared to the original sfGFP

By doing this we improved sfGFP (by expanding the range of possible excitation/emission optima). One could 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 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, display high internal fluorescence, which make impossible to visualize sfBFP in liquid without significant 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 fluorescence levels of sfGFP (the fluorescence intensity is normalized, hence the most potent fluorescent protein has 100% fluorescence ).

Tab 1. – Fluorescence 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 maxima: 503/540 nm.

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

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. Comparison with sfVenus (and sfCerulean) protein which we also designed this year would be most interesting. Unfortunately, the synthetic construct did not reach us in time before wiki freeze.

We created superfolder BFP, CFP and YFP with the intention of creating a “BiFC Toolbox” for synthetic biology. 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 truncated 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 also work well with other fluorescent proteins as well. C-terminal is in two variants: m6 and m12 (differing in sensitivity to specificity ratio). Moreover, we attempted to produce BiFC fragments from 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 those parts to Parts Registry. C-m6 and C-12 fragments due in part to their small size, proved difficult to clone. Thus, we didn’t manage to submit the DNA samples before the deadline.

To verify the functionality of our BiFC fragments, we decided to use b-Fos and b-Jun proteins, that interacts with each other. We planned to fuse b-Jun with N-terminal fragment and b-Fos with C-terminal one. 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. For measurements we lysed bacteria expressing either b-Fos or b-Jun fused with part of fluorescent protein and mixed the lysates. This way we will create not only the wide range of BIFC proteins of different parameters, but also provide systematic and standardized information about sensitivity and specificity of the each combination. We will also measure Venus- and Cerulean-based BIFC fragments already present in the registry.

Unfortunately, vacation is over and we did not manage to fulfill our ultimate goal and measure our constructs this year. We will however continue working on the toolbox, as we feel it will prove a valuable addition to the Registry.

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.