The importance of controls, proper annotation, and methodically working through unexpected results…

For one of my projects I prepared the turbo Green Fluorescent Protein molecule we have in our Golden Gate parts collection and installed this into a plasmid (pUC57-Kan) containing a fragment I had synthesized. My goal was to produce an inducible scorable marker (which should be possible with the lacZ promoter) to easily identify plasmids which contain the tGFP sequence in the targeted interval. I spread some of the transformation recovery broth on a solid LB plate supplemented with kanamycin, IPTG (to induce the lacZ promoter), and X-gal (ordinarily utilized as the substrate for blue/white selection in E. coli, although there shouldn’t be a β-galactosidase in my plasmids, but the plates were already in the refrigerator). I decided to include an uninduced control plate and delivered a portion of the recovery broth to a plate supplemented with kanamycin (no IPTG or X-gal).

Since my synthesized fragment does not contain a β-galactosidase, I was quite surprised to see blue colonies on the induction plate. I became concerned since my project plan includes using for a different region of the plasmid  blue/white selection via the lacZ promoter driven β-galactosidase . It seemed like the GFP expression might really be higher in cells taken from the induction plate (in the upper most micrograph there was no GFP visible in cells containing a plasmid which does not contain the GFP gene), but the blue color was completely unexpected. I really needed to figure out what was going on.

After a bike ride and a shower it occurred to me that I had unwittingly fooled myself with my typical plasmid annotation process. When I receive plasmid sequences from the synthesis company, I usually start the annotation process by first opening the molecule file in SnapGene viewer, and asking the program to find features. This can be helpful, but in this instance, the program did not “see” the partial β-galactosidase remaining in the pUC57-Kan plasmid after the synthesis company had installed my synthetic piece. The original β-galactosidase sequence in pUC57-Kan is 345 basepairs (uppermost cartoon), resulting in a protein of 115 amino acid residues. The synthesized fragment in pUC57-Kan results in the elimination of a portion of the original β-galactosidase sequence, but an open reading frame of 273 basepairs remains (shown as a black arrow in the middle cartoon, as well as the black arrow downstream of the tGFP in the lower cartoon) that could produce a partial β-galactosidase of 91 amino acid residues.

Alignment of the two protein sequences reveals that there is a methionine in the modified version that can act as a start codon for the partial β-galactosidase. In spite of the highly modified N terminus (with a deletion of 22 amino acids, as well as 7 non-silent mutations) the modified β-galactosidase protein, which is in frame with the tGFP, is sufficiently functional to cleave the X-gal substrate to produce the blue precipitant. Fortunately, the tGFP modified fragment will be liberated from this initial construct by SapI digestion, and the destination plasmid does not contain a β-galactosidase gene, but I will for sure continue to include proper controls when building the next construct in this series.