DNA Construct Benchling

Design 01 : Substitution within Template

Following the steps demonstrated during recitation Week 02, I cut the sfGFP insert from the plasmid template ColE1-AmpR-sfGFP and replaced it with the optimized firefly luciferase sequence (including flanks 5’ and 3’).

Step 01: Load plasmid template ColE1-AmpR-sfGFP in Benchling

Step 02: Copy plasmid template ColE1-AmpR-sfGFP into a new project page, cut sfGFP and paste the optimized luciferase sequence.

Step 03: Run a digest of the entire construct to verify that the MluI and AatII restrcition enzymes (REs) specifically cut it within the restriction sites domains. i.e. flanks 5’ (A | CGCGT cut) and 3’(GACGT | C cut).

RESULT: Fail. The digest looked promising (2 bands of different lengths), but when checking the bp values, the numbers (~4 kb and ~150 bp) did not match the construct. Zooming in on flanks 5’ and 3’, I realized that flank 3’ did not contain the AatII restriction site, as Claude had claimed.

Presence of MluI in Flank 5’ validated

Presence of AatII in Flank 3’ not confirmed

Lesson learned Double-check every single AI suggestion and strengthen the prompts.

Step 04: Fast troubleshooting. (1) Exchange one base (C instead of T in position 1'739) to create AatII restrcition site within flank 3’. (2) Exchange one base (C instead of T in position 4'286) to remove MluI restrcition site in another part of the plasmid.

RESULT: Success. One can see on the digest one band corresponding to the backbone vector (2683bp) and another one, lower, corresponding to the Firefly Luciferase insert (1672 pb).

Final Step: Experimental validation of the DNA construct to determine whether it leads to the production of luciferase or not.