cn mdh2 ct4 1 mp66 addgene  (Addgene inc)

Journal: ACS synthetic biology

Article Title: Multiple Rounds of In Vivo Random Mutagenesis and Selection in Vibrio natriegens Result in Substantial Increases in REE Binding Capacity.

doi: 10.1021/acssynbio.3c00484

Figure Lengend Snippet: Figure 1. Induction of the MP6 plasmid increased the DNA mutation rate in the fast-growing microbe Vibrio natriegens and produced strains with higher REE biosorption. (A) Induction of MP6 with 100 mM arabinose increased the mutation rate by 2200 relative to the wild-type without MP6. Some leaky expression caused the mutation rate in V. natriegens to increase by a factor of 8 without MP6 induction. (B) Arsenazo-III (As-III) is a metallochromic dye that changes from a pinkish to a blue-greenish color (with an absorption peak at 660 nm) when it binds to lanthanides (most of the REE). (C) We mutagenized a culture of V. natriegens by inducing MP6, plated the culture on agar, robotically picked colonies into 96-well plates, and then measured the amount of dysprosium biosorption by each mutant strain with the As-III assay. The absorbance at 660 nm indicates how many REE remained in solutionthe amount of REE that was not biosorbed. We compared the As-III-Dy absorbance with the amount of bacteria in solution (through the optical density of the culture at 590 nm) since the more the bacteria, the more REE would be adsorbed. We plotted a line of best fit by comparing the 660 nm absorbance and the optical density. We picked the outlier from each round of mutagenesis with the highest biosorption to use in further rounds of mutagenesis. (D) Three rounds of mutagenesis with MP6 improved Dy biosorption by up to 210% (R3n3). For rounds one and two, we picked one mutant (R1 and R2, respectively), while for round three we picked three mutants (labeled R3n1, R3n2, and R3n3). All changes relative to the wild-type were statistically significant (p-value < 0.05).

Article Snippet: The MP6 plasmid was ordered from AddGene (#69669) and its sequence was confirmed through whole plasmid sequencing by Plasmidsaurus.

Techniques: Plasmid Preparation, Mutagenesis, Produced, Expressing, Bacteria, Labeling

Journal: ACS synthetic biology

Article Title: Multiple Rounds of In Vivo Random Mutagenesis and Selection in Vibrio natriegens Result in Substantial Increases in REE Binding Capacity.

doi: 10.1021/acssynbio.3c00484

Figure Lengend Snippet: Figure 5. Spectrum of genomic mutations induced by MP6. (A) Most genetic changes that we observe are transition mutations (i.e., a pyrimidine to a pyrimidine or a purine to a purine). (B) We compared the number of silent and nonsilent genomic changes in each strain compared to the previous strain. We defined nonsilent mutations as genetic changes that alter an amino acid in a coding region or that occur within 100 bp of the beginning of the gene. All other mutations were considered silent.

Article Snippet: The MP6 plasmid was ordered from AddGene (#69669) and its sequence was confirmed through whole plasmid sequencing by Plasmidsaurus.

Techniques: