two or three GFPs (or any other fluorophore) in tandem in worms?

Model Organism: Worms Scientific Discussion
1

1- Has anyone generated two or three GFPs (or any other fluorophore) in tandem in worms?
2- Has anyone generated a combined protein of GFP + a variant of GFP (let’s say five tandem of pH-sensitive GFP): to have a GFP that can change color or gets brighter (not in lysosomes) for example, and can be tracked.

I’m using traditional extrachromosomal array and working with a gene that cannot be overexpressed, therefore I am injecting it with the lowest concentration 1ng/ul. I am facing a very weak fluorescence.
Seeing papers in other systems (links below), I am thinking to insert additional GFP after the GFP:
promoter::gene:: GFP-linker-GFP-linker-GFP
promoter::gene:: GFP-linker-GFP variant

I would be grateful if you can help me to understand if this is a good idea and I don’t get GFP polymerization or perhaps protein of interest misfolding.

Papers:
- Hexameric GFP and mCherry Reporters for theDrosophilaGAL4, Q, and LexATranscription Systems

… To augment the ability to visualize cells or tissues of interest in Drosophila melanogaster, homo-hexameric GFP and mCherry reporters were developed … The GFP and mCherry homo-hexameric fusion proteins exhibited significantly enhanced fluorescence as compared to monomeric fluorescent

- Recombination-stable multimeric green fluorescent protein for characterization of weak promoter outputs in Saccharomyces cerevisiae

…Unlike a single GFP, the brightness of 3vGFP allowed characterization of a weak promoter in S. cerevisiae.

… We increased GFP fluorescence by translationally fusing three different GFP variants, yeast-enhanced GFP, GFP+ and superfolder GFP to yield a sequence-diverged triple GFP molecule 3vGFP with 74-84% internal repeat identity…

- Tandem fluorescent protein timers for in vivo analysis of protein dynamics

2

At one point when I was a postdoc, I made a 4x GFP construct and integrated with MosSCI. It worked, but the GFPs recombined with each other during the MosSCI insertion; from a plasmid encoding 4x GFP, I isolated lines carrying 0, 1, 2, 3, 4 and 5(!) tandem copies of GFP. This could be either convenient (eliminates the need to clone several constructs) or a total pain (requires some work to sort out which lines are which). Depends on what you’re after.

I would suspect that with extrachromasomal arrays or any multicopy scenario, the recombination problem would be significantly worse, and you may never know what you have.

One of lab mates in the Goldstein lab made several 2xGFP or 2x mTurquoise lines, and eliminated the recombination issue by using different codons for the two copies of the FP. This seems like a good solution and worked reproducibly for several lines, as I recall.

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Thanks. I’m actually thinking to use this strategy for other FPs. But, first 2x mEGFP as a control. One of the papers used YFP+GFP.

I may ask Goldstein lab.

4

There are several linker sequences around, but I am not sure which one is a good one to put between two or perhaps three Fluorophores.

May I ask you what nucleotide sequence (or amino acids) you are using as a linker?