Part:BBa_K4830028

eGFP_simple premature stop codon

eGFP_premature stop codon is a protein coding sequence that contains the eGFP with a premature stop codon. This coding sequence is particularly useful for Traffic Light Reporter (TLR) assays that screen for DNA editing activity. pegRNA and ngRNA targeting this particular sequence have already been designed - PETLR pegRNA 1 and PETLR ngRNA 1

Usage and Biology

This sequence is used in a TLR assay for prime editing where pegRNAs and ngRNAs targeting the premature stop codon were designed to swap the premature stop codon into a functional amino acid, restoring the wild-type eGFP.

When cloned into a suitable plasmid and subsequently transfected into mammalian cells for in vitro DNA editing, successfully edited cells will fluorescence green which can then be analysed with flow cytometry.

Characterization

The pegRNA in combination with ngRNA were used to test the efficiency of the Prime Editor containing the alternative Reverse Trancriptases. The pegRNA served as template to install the edit. 3 plasmids containing PE, pegRNA and ngRNA were trasfected into HEK293T cells, and editing efficiency was evaluated 72hr after transfection using flow cytometry by evaluating either the mean fluorescence intensity of the enhanced green fluorescent protein (eGFP), or the percentage of cells containing the eGFP.

PETLR pegRNA 1 (BBa_K4830023) was co-transfected with PETLR ngRNA 1 (BBa_K4830024). The targeting sequence was the premature stop codon found on the eGFP requiring a simple edit (BBa_K4830028).

Fig. 1 Bar graph shows the mean P2 FITC-A from the three replicates, and scatter points denote the individual P2 FITC-A from each replicate. P2 FITC-A is a measurement of the mean fluorescence intensity of the eGFP.

Sequence and Features

The sequence contains the eGFP with a premature stop codon.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]