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  1. In literature, Nocardia cholesterolicum NRRL 5767 (NC NRRL5767) is well-known for its ability to transform ~95% of added oleic acid, an abundant agricultural commodity, to value-added product of 10-hydroxystearic acid (10-HSA). A small amount of unwanted 10-ketostearic acid (10-KSA) was also produced. This microbe also transforms ~80% of added linoleic acid to 10-hydroxy-12(Z)-octadecenoic acid (10-OH-12-OD) (an isomer of ricinoleic acid) with minor 10-oxo-12(Z)-octadecenoic acid (10-oxo-12-OD). The conversion of oleic acid to 10-HSA and then to 10-KSA (or linoleic acid to 10-OH-12-OD and then to 10-oxo-12-OD) is catalyzed by oleate hydratase and secondary alcohol dehydrogenase (2o-ADH), respectively. The objective of thismore »project was to knockout the 2o-ADH gene in NC NRRL5767 so that the sole biotransformation product from oleic acid would be 10-HSA. Here, we report construction of CRISPR/Cas9/sgRNA chimeric plasmid that specifically target 5’ coding region of the 2o-ADH gene by Golden Gate Assembly. The construct was confirmed by DNA sequencing and transformed into NC NRRL 5767 via electroporation. The transformants were selected by apramycin resistance and screened for the presence of the target insert (crRNA) by PCR. The ability of the selected transformants to transform oleic acid to 10-HSA was screened by TLC and further confirmed by GC-MS. Our results showed that two of the transformants produced only 10-HSA with no detectable 10-KSA from oleic acid suggesting successful knockout of the 2o-ADH gene. Final confirmation came from the isolation of genomic DNA from these two transformants and the wild type NC NRRL5767 (used as DNA template) and using 17 primers (locate at different positions along the 2o-ADH gene and the 5’ upstream of this gene) for PCR. To our best knowledge, this is the first report to knockout the target gene in Nocardia species by CRISPR-Cas9 technology.« less
  2. Free, publicly-accessible full text available March 1, 2022
  3. Abstract The prediction of reactor antineutrino spectra will play a crucial role as reactor experiments enter the precision era. The positron energy spectrum of 3.5 million antineutrino inverse beta decay reactions observed by the Daya Bay experiment, in combination with the fission rates of fissile isotopes in the reactor, is used to extract the positron energy spectra resulting from the fission of specific isotopes. This information can be used to produce a precise, data-based prediction of the antineutrino energy spectrum in other reactor antineutrino experiments with different fission fractions than Daya Bay. The positron energy spectra are unfolded to obtainmore »the antineutrino energy spectra by removing the contribution from detector response with the Wiener-SVD unfolding method. Consistent results are obtained with other unfolding methods. A technique to construct a data-based prediction of the reactor antineutrino energy spectrum is proposed and investigated. Given the reactor fission fractions, the technique can predict the energy spectrum to a 2% precision. In addition, we illustrate how to perform a rigorous comparison between the unfolded antineutrino spectrum and a theoretical model prediction that avoids the input model bias of the unfolding method.« less
    Free, publicly-accessible full text available July 1, 2022
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