Plasmid-based experiments have revolutionized biological research. Scientists use this technique to induce and silence the expression of specific proteins in in vitro and in vivo models as they investigate the mechanisms underlying health and disease.

Plasmids comprise small circular DNA molecules that can be molecularly engineered to express a specific gene. Host cells receive several copies of the plasmid through transfection and then use their own metabolic machinery to transcribe the gene of interest. In some experiments, the final molecule is a protein or enzyme. In others, plasmids encode small interfering RNA (siRNA) or short hairpin RNA (shRNA), which then silence the expression of other genes.

Scientists can use plasmid-based experiments in many ways to answer various research questions. But without proper controls, acquired data will be inconclusive and unreliable. This blog answers frequently asked questions about selecting appropriate controls, so you can confidently design your next plasmid-based experiment. It also highlights how the eZ-stop peptide can simplify the process and provide an effective solution for all your experimental control needs.

Why is it essential to select proper controls for plasmid-based experiments?

The selection of proper controls ensures the validity and reliability of acquired results. Transfection is not a silent process and can, by itself, trigger cellular stress responses and changes in gene expression. Adding proper controls to plasmid-based experiments can help ensure that observed changes result from the manipulated gene expression and no other variables. In addition, appropriate controls can help answer the following questions:

• Did the transfection work?
• Did the experimental procedure induce cytotoxicity?
• How efficient was the transfection process?
• Can the cells translate and transcribe the selected plasmid?
• Why didn’t the experiment work?

What are the types of controls frequently used in plasmid-based experiments?

When performing plasmid-based experiments, you should consider 2 types of controls:

• Positive controls make assessing whether the transfection worked and whether the host cell can translate and transcribe the selected plasmid easier.
• Negative controls mimic as much as possible all factors present in the experimental condition—transfection, reagent, plasmid (not exhaustive)— without the expression of gene(s) of interest and are designed to confirm that any phenotypical change observed post-transfection is actually dependent on the gene of interest and not an artefact due to the experimental process.

What positive controls are commonly used in plasmid-based experiments?

Positive controls comprise the same vector used in the experimental condition expressing a gene unrelated to the gene of interest. Adding positive controls to your experimental design can help you assess the efficiency of transfection, transcription and translation. You can create a plasmid expressing reporter genes such as GFP and luciferase and easily detect whether transfection and transcription worked. Alternatively, you can use housekeeping genes, such as GAPDH and quantify protein expression as a measure of success. In gene knockout experiments, you can use positive controls that will silence the expression of a gene unrelated to the experimental condition. It’s important to highlight that the gene silenced in the positive control condition should not be essential to cell survival. You can co-transfect non-specific plasmids with the plasmid of interest or use a different pool of cells to express the positive control gene.

What types of negative controls should I consider for my experiments?

The primary purpose of negative controls is to assess the impact, the transfection and transcription of foreign DNA have on cell function, metabolism, and toxicity. You can use an empty plasmid —the same genetic backbone without the gene of interest—as a negative control. But this approach imposes a different metabolic on the host cell. Using empty plasmids as negative controls is technically convenient but has many flaws. Empty plasmids are smaller, have a different transfection rate, and impose a lower metabolic burden on the host cell than plasmids containing the gene of interest. In addition, empty plasmids don’t yield any transcripts; thus, you can’t control for RNA interaction when using this approach.

One alternative is to use mutant plasmid controls, where the same vector from the experimental condition expresses a mutant version of the gene of interest lacking functionality. When choosing this approach, you should match the length and C/G content of the mutant gene to those of the gene of interest. In addition, the sequence of the mutant gene should not interfere with mRNA translation.

Creating your own mutant plasmid control is time consuming as it requires additional engineering steps to redesign or edit the gene of interest sequence. But you can streamline this step by using a pre-designed sequence called eZ-stop, which is a better approach and is a superior negative control than empty plasmids. When using the eZ-stop peptide, final experimental and negative control plasmids have similar lengths, resulting in similar transfection rates and metabolic burden (Figure 1). In addition, both experimental and negative control conditions trigger the transcription of the gene of interest. In summary, using the eZ-stop peptide helps you isolate the effect of the independent variable with more precision. As a result, data validity and reliability improve.

Figure 1. Comparison of empty control plasmid and eZ-STOP control plasmid.

What’s the eZ-stop peptide?

The eZ-stop peptide is a 27-nucleotide long sequence that you can insert between the ATG and the second codon of the coding sequence. It encodes a hexapeptide (GGSIIR) followed by 3 stop codons: TAA ‘ochre,’ TAG ‘amber,’ and TGA ‘opal’ in phase with the initiation codon (ATG) (Figure 2). Stop codons are intentionally placed 18 nucleotides apart from the ATG to minimize the risk of readthrough by translating ribosomes. The eZ-stop peptide also contains stop codons in frames 2 (TAA) and 3 (TAG) to avoid the translation of products that could be transcribed from cryptic initiating sequences that may occur in the plasmid (Figure 2). Using the eZ-stop peptide makes it easier to generate mutant plasmid controls.

Figure2. eZ-STOP peptide sequence

How effective is the eZ-stop peptide in silencing the expression of the gene of interest?

Quality control experiments revealed that adding the eZ-stop peptide into different plasmids inhibited the expression of the gene of interest with more than 99% efficiency. In these experiments, the eZ-stop peptide was added to plasmids with promoters of various strengths—pmPGK, pCMV, pEF1a—expressing eGFP. HEK-293-T cells were transfected with final constructs, and eGFP expression was analyzed 48 hours post-transfection. As expected, different promoters expressed varied levels of eGFP with the pEF1a promoter showing the highest strength, followed by pCMV and pmPGK (Figure 3). But the insertion of the eZ-stop peptide effectively inhibited eGFP expression regardless of the promoter strength (Figure 3).

Figure 3. eZ-STOP peptide can efficiently block gene expression induced by promoters of different strengths

What are some final tips for using the eZ-stop peptide?

You can use the eZ-stop peptide in any expression vector. You can also insert it between protein domains to obtain a 3’ truncation of the protein of interest. For example, this approach would be helpful when creating controls for FRET experiments. In addition, inserting the eZ-stop peptide downstream into a reporter gene can help you test the promoter activity without expressing the protein of interest.

Discover eZ-stop peptide and additional features on our plasmid drawing online platform.

• Design your plasmids by relying on a fully flexible tool. Just like Lego, you have complete freedom to move each sequence as you want within a plasmid.
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• Have your design reviewed and validated by plasmid design experts. Or have our plasmid experts help you from the start of design.

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Reach out to the technical support team to request a modified sequence adapted to the codon usage of unusual biological models.