Part:BBa_K4830029

eGFP_complex premature stop codon

The eGFP_complex premature stop codon is a derivative of the eGFP_simple premature stop codon. Similar to the eGFP_simple premature stop codon, the eGFP_complex premature stop codon can be utilized in a Traffic Light Reporter (TLR) assay.

However, the edit required is complex as it involves the truncation in a particular region from 42bp to 31bp. Specific pegRNA and ngRNA targeting the premature stop codon have been designed - TLR-76 pegRNA and 777BFP ngRNA.

Usage and Biology

The eGFP_complex premature stop codon is a coding sequence used in the Traffic Light Reporter (TLR) assay. It contains a particular region that requires a complex edit to truncate from 42bp to 31bp, before restoring the wild-type eGFP.

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 enhanced green fluorescent protein (eGFP), or the percentage of cells containing the eGFP.

TLR-76 pegRNA (BBa_K4830025) was co-transfected with 777BFP ngRNA (BBa_K4830026). The targeting sequence was the premature stop codon found on the eGFP requiring a complex edit (BBa_K4830029). Initial testing were performed on two different cell lines containing the integrated sequence. HEK293T TLR WT is a heterogeneous cell line containing the integrated sequence, while HEK293T 1D10 is a homogeneous cell line derived from the former.

Fig. 1 Bar graph shows the P3 FITC-H Mean (indicating the mean fluorescence intensity of eGFP), and scatter points denote the P3% Parent (indicating the percentage of cell containing correctly edited wild-type eGFP). Results from initial testing with TLR WT cell line.

Fig. 2 Bar graph shows the P3 FITC-H Mean (indicating the mean fluorescence intensity of eGFP), and scatter points denote the P3% Parent (indicating the percentage of cell containing correctly edited wild-type eGFP). Results from initial testing with TLR 1D10 cell line.

Sequence and Features

The coding sequence spans the eGFP with an inherent premature stop codon to be edited.

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 192
  Illegal PstI site found at 160
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 192
  Illegal PstI site found at 160
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 192
  Illegal PstI site found at 160
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 192
  Illegal PstI site found at 160
  Illegal NgoMIV site found at 734
  
• 1000
  COMPATIBLE WITH RFC[1000]