Transition Between Supercoiled and Open Circular Plasmid DNA During Alcohol Precipitation

Tomanee, Panarat; Hsu, Jong-Ping

doi:10.1081/jlc-120034086

Cited by 2 publications

(4 citation statements)

References 8 publications

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Section: Resultsmentioning

confidence: 99%

“…Interestingly, the sfGFP expression was not affected by the lack of extra purification steps, but mNG was significantly affected, with an ∼70% decrease, even after repeating the experiment multiple times (Figure C). Additional isopropanol precipitation in the last step of midi and maxiprep purification may affect the plasmid structure transition between supercoil and circular in different DNA sequences and eventually affect the overall protein production level difference between sfGFP and mNG …”

Section: Resultsmentioning

confidence: 99%

“…Additional isopropanol precipitation in the last step of midi and maxiprep purification may affect the plasmid structure transition between supercoil and circular in different DNA sequences and eventually affect the overall protein production level difference between sfGFP and mNG. 64 The maximum concentration of plasmid DNA for the maximum amount of protein has been shown to plateau at 5− 15 nM of DNA concentration when using endogenous RNA polymerase for transcription. In another study, however, the DNA concentration of plasmid and LET was not reflected in the overall transcription and translation efficiency using T7 RNA polymerase.…”

Section: T H Imentioning

confidence: 99%

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Characterizing a New Fluorescent Protein for a Low Limit of Detection Sensing in the Cell-Free System

et al. 2022

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Cell-free protein synthesis-based biosensors have been developed as highly accurate, low-cost biosensors. However, since most biomarkers exist at low concentrations in various types of biopsies, the biosensor’s dynamic range must be increased in the system to achieve low limits of detection necessary while deciphering from higher background signals. Many attempts to increase the dynamic range have relied on amplifying the input signal from the analyte, which can lead to complications of false positives. In this study, we aimed to increase the protein synthesis capability of the cell-free protein synthesis system and the output signal of the reporter protein to achieve a lower limit of detection. We utilized a new fluorescent protein, mNeonGreen, which produces a higher output than those commonly used in cell-free biosensors. Optimizations of DNA sequence and the subsequent cell-free protein synthesis reaction conditions allowed characterizing protein expression variability by given DNA template types, reaction environment, and storage additives that cause the greatest time constraint on designing the cell-free biosensor. Finally, we characterized the fluorescence kinetics of mNeonGreen compared to the commonly used reporter protein, superfolder green fluorescent protein. We expect that this finely tuned cell-free protein synthesis platform with the new reporter protein can be used with sophisticated synthetic gene circuitry networks to increase the dynamic range of a cell-free biosensor to reach lower detection limits and reduce the false-positive proportion.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: T H Imentioning

confidence: 99%

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Characterizing a New Fluorescent Protein for a Low Limit of Detection Sensing in the Cell-Free System

et al. 2022

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“…It is well known that chromosomal DNA may have different degrees of supercoiling and that plasmids can exist in linear, relaxed-circular, or supercoiled forms. Because essential cellular processes, such as DNA packaging, transcription, replication, recombination, response to environmental stress, and protein binding are all dependent on DNA topology [2][3][4][5][6][7][8], a simple method that can simultaneously determine DNA conformation and base number would be advantageous.…”

Section: Introductionmentioning

confidence: 99%

DNA conformation and base number simultaneously determined in a nanopore

et al. 2007

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When dsDNA polymers containing the identical number of base pairs were electrophoresed through a nanopore in a voltage biased silicon nitride membrane, the measured time integral of blocked ionic current (the event charge deficit, ecd [1]) for each translocation event was the same regardless of whether the molecules were in a linear, circular relaxed, or supercoiled form. Conversely, when DNA polymers containing different numbers of base pairs were electrophoresed through a nanopore, the ecd depended strongly on, and predicted the value of, the molecule's number of base pairs. Measurements showed that the magnitude of the current blockages was strongly affected by a molecule's form. The current blockages exhibited characteristic differences that distinguished between single stranded linear, double stranded linear, circular relaxed, and supercoiled forms. Because the data that establish ecd are usually determined concomitantly with current blockade measurements, our results show that a single nanopore assay can simultaneously determine both DNA conformation and base number.

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Transition Between Supercoiled and Open Circular Plasmid DNA During Alcohol Precipitation

Cited by 2 publications

References 8 publications

Characterizing a New Fluorescent Protein for a Low Limit of Detection Sensing in the Cell-Free System

Characterizing a New Fluorescent Protein for a Low Limit of Detection Sensing in the Cell-Free System

DNA conformation and base number simultaneously determined in a nanopore

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