AI Article Synopsis
- Inactivation of the TK1761 gene revealed a second beta-glycosidase enzyme produced by TK1827, which functions effectively at high temperatures and specific pH levels.
- A new selection protocol has been developed to delete both TK1761 and TK1827 in the Thermococcus kodakarensis strain, allowing precise genetic manipulation.
- The technology also enables the deletion of nonessential genes, demonstrated by creating a strain that requires added agmatine for growth in a nutrient-rich medium.
Article Abstract
Inactivation of TK1761, the reporter gene established for Thermococcus kodakarensis, revealed the presence of a second beta-glycosidase that we have identified as the product of TK1827. This enzyme (pTK1827) has been purified and shown to hydrolyze glucopyranoside but not mannopyranoside, have optimal activity at 95 degrees C and from pH 8 to 9.5, and have a functional half-life of approximately 7 min at 100 degrees C. To generate a strain with both TK1761 and TK1827 deleted, a new selection/counterselection protocol has been developed, and the levels of beta-glycosidase activity in T. kodakarensis strains with TK1761 and/or TK1827 deleted and with these genes expressed from heterologous promoters are described. Genetic tools and strains have been developed that extend the use of this selection/counterselection procedure to delete any nonessential gene from the T. kodakarensis chromosome. Using this technology, TK0149 was deleted to obtain an agmatine auxotroph that grows on nutrient-rich medium only when agmatine is added. Transformants can therefore be selected rapidly, and replicating plasmids can be maintained in this strain growing in rich medium by complementation of the TK0149 deletion.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2820962 | PMC |
| http://dx.doi.org/10.1128/AEM.02497-09 | DOI Listing |
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