I am trying to insert two mutations in a gene that we have used before. The original synthetic gene was purchased in a pUC57 plasmid and then we cloned it in a pET28a(+). E. coli transformed did not showed any growth issues.

I performed PCR in order to obtain the original gene (from synthetic plasmid pUC57) and at the same time modify one of the restriction sites (allows cloning in pET28a(+) with BamHI and EcoRI restriction sites). This also we have done before.

Once I obtained the original gene, I performed overlap extention PCR using the oligonucleotides mentioned and the oligonucleotides designed to insert the mutations. After those rounds of PCR I obtained a DNA fragment (gene) with the correct size and a good amount of it (2% agarose gel).

Then, restriction assay was performed (Bam HI - Eco RI) with pET28a(+) and "my gene". After digestion, gel purification, and cuantification I performed a T4 DNA ligase (Thermo Scientific) assay. I have tried different temperatures, insert:plasmid ratios, and ligation times. E. coli Top 10 or B10 cells transformed with the plasmid pET28a(+)-mutgene show normal growth on LB solid media + 50 ug/mL kanamycin. Colonies selected were grown in liquid LB-50 ug/mL kanamycin at 37 C and 150 rpm shaking for 16-18 h. These parameters were used with E. coli BL21 (DE3) transformed with pET28a(+)-original-gene and no issues were observed. But E. coli transformed with the plasmid with the mutant gene show an abnormal growth. The liquid LB medium does not turn opaque insted it keeps its transparency but with bacteria dots sprayed everywhere. Finally, plasmid DNA extraction methods have been done and no plasmid have been found.

What could I be doing wrong? Is there anyone who has had the same problem? Any advise is welcome.

Thanks.

NOTE 1: Top 10 and B10 cells transformed with pET28a(+) grow normally.

NOTE 2: When neccesary PCR purification assay was performed.