Journal: Heliyon
Article Title: Markerless bacterial artificial chromosome manipulation method by red proteins of phage λ mediated homologous recombination utilizing fluorescent proteins for both positive and counter selection
doi: 10.1016/j.heliyon.2023.e18983
Figure Lengend Snippet: Optimization of template plasmids. pAmp-9mScarlet without the terminator rrnB T1 was constructed (A). mScarlet protein expression in the pelleted HTS16CR cells was observed on an LED transilluminator after the cells were transformed with pAmp-9mScarlet (+) or pAmp-9mScarlet without a terminator (−) following overnight cultivation in 2 mL of LB medium at 37 °C (B) . Additionally, the plasmid yields of 2 mL of HST16CR cultures transformed with pAmp-9mScarlet (+) (with terminator) and pAmp-9mScarlet (−) (without terminator) were compared (C). The fluorescent protein gene (FP), EGFP, mClover3, YPet, mOrange, mKO2, or mScarlet were ligated into a plasmid (D), which has the same backbone as pAmp-9mScarlet, except for the fluorescent gene, as shown in . E. coli , HST16CR, or HST08 were transformed with these plasmids or pAmp-S as the negative control (control) and then cultivated on LB agar plates at either 30 °C or 37 °C overnight under ampicillin selective pressure (E) (Supplementary fig. 3E-1, -2 and -3). Note that HST08 cells transformed with mKO2 did not grow on the plates either at 30 °C or 37 °C. An unpaired t -test was used to determine the statistical significance. The calculated p values are shown above the compared groups.
Article Snippet: The mKO2 gene was obtained from mKO2-pBAD gifted by Michael Davidson and Atsushi Miyawaki (#54555, Addgene) [ ].
Techniques: Construct, Expressing, Transformation Assay, Plasmid Preparation, Negative Control, Control
