eZ-Stop Peptide: better negative controls in your experimentations.
What was done up to date?
When it comes to setting up your transgenesis experimentation, using either eukaryotic or prokaryotic cells, one would always ponder the options for negative control to include. Because the introduction of the vector (plasmid) to the cell (e.g. by transfection or transformation) is not a silent process, physiologically speaking, on accepted good practice is to monitor what happens when cells are treated exactly the same way that for the test condition, without the possibility of expressing the gene of interest, but transferring a vector that will not express the gene of interest. Such a condition is sometimes described as ‘mock’ or negative control, and the vector that is used to create it should be the closest to the test vector as possible to limit the biological biases. Most of the time, scientists working with classical expression vectors, would thus choose to use the same vector backbone, without the insertion of their gene of interest, referred as ‘empty vector’. More rarely, they would insert another gene that is expected to not affect the experimental readout and that can be used as a reporter gene for technical control.
What was done up to date?
At e-Zyvec, we believe that the traditional ‘empty vector’ used as negative control is not entirely satisfactory. A number of biases are currently disregarded (figure 1), mostly because using empty vector backbones is technically convenient.
Figure 1: Pros and Cons of eZ-Stop control vectors compared to empty vector.
The eZ-stop peptide: simple yet elaborate...
We thought that a better negative control should be an expression vector that would trigger the transcription of the gene of interest without allowing its translation. Thus, the modified cells would behave exactly the same, metabolically, until the initiation of the translation and then the protein synthesis would be aborted.
To achieve this, we designed a 27 nucleotides long sequence (Figure 2), that is to be inserted between the ATG and the second codon of the coding sequence. This sequence encodes an hexapeptide (GGSIIR) followed by three stop codons: TAA ‘ochre’, TAG ‘amber’ and TGA ‘opal’ in phase with the initiation codon (ATG). We voluntarily placed the stop codons 18 nucleotides apart from the ATG to minimize the risk of readthrough by translating ribosomes (CF ez-101: Protein translation).
Figure 2: eZ-stop coding sequence showing the starting codon in green, the hexapeptide in purple, and frame 1 stop codon in red. Translation fram e1, 2 and 3 are presented below the nucleotide sequence, showing stop codons in every frame as red-boxed asterisks.
On top of this, we also included stop codons in frame 2 (TAA) and in frame 3 (TAG) to avoid any other translation of alternate products that could be transcribed and translated from cryptic initiating sequences that may occur in the plasmid.
... And efficient!
The eZ-stop sequence was introduced in some of our PromeZ vectors to assess its efficiency to block protein expression from a full size vector. We chose to work with three constitutive promoters of various strength in our model (HEK-293-T), namely pmPGK < pCMV < pEF1α. The figure 3 below shows that eZ-stop sequence is inhibiting the eGFP expression in transfected cells by more than 99%, independently of the strength of the tested promoter.
Figure 3: eGFP expression measured by fluorescence detection using a plate reader. Briefly, 3.104 HEK-293-T cells were transfected with eGFP encoding plasmids containing or not the eZ-Stop sequence and analyzed 48hours post-transfection. Each condition was measured in three independent wells and the graph is showing the average of 3 independent transfection experiments.
eZ-STOP tips and usage:
The eZ-stop sequence can be inserted within any expression vector you wish to get a proper negative control for.
We can even insert it between protein domains to obtain 3’ truncation of your proteins of interest (FRET control anybody?) or downstream a reporter gene, so you can still demonstrate your promoter activity, while lacking the protein of interest.
And if you are working on unusual biological model, we can certainly look into adapting the sequence to its codon usage.