They inserted the cassette into a circular piece of DNA called a plasmid and allowed it to be taken up by another bacterium, Escherichia coli, which then started using the foreign genes, an indication that they had inserted into E. coli's own integron.
To test the efficiency of inserting our STOP-dsRed cassette, we designed three donor constructs targeting different regions in the salm gene: the first, deleting parts of exon 1; the second, inserting the cassette into intron 1; and the third, deleting exon 3.
A) Expression vector pET21a used as backbone to insert the expression cassette; B) Expression cassette comprised of a signal sequence (Ss), a HIS-tag, the thioredoxin gene end the ClavMO peptide.
Some Destination vectors insert the Entry cassette in their 3'LTR (see below), but this imposes an insert size limit, usually ∼800 bp [37], because larger inserts inactivate the LTR and impair viral integration.
When the products were analyzed by Type II BESt™ cassette, the melt curve analysis were not performed, instead the reaction tubes were inserted to the cassette for specific detection of target and/or internal control amplification.
After transformation of the donor plasmid carrying the synthetic DNA segment into the strain containing the landing pad inserted genome, the cassette exchange was induced by galactose for 24 hours, followed by selection for uracil protorophy.
Sequential rounds of insertional gene targeting were used to insert the selection cassettes and loxP sites required for chromosome engineering proximal to the Herc2 and distal to Mkrn3.
Using the same strategy as the pLenti X2 series, we inserted the Gateway Destination cassette into the 3' LTR of the retroviral backbone so we could obtain the expression of two shRNA/miRNA cassettes upon viral integration into the cellular genome (pQCXI X2 series, Figure 3F, 4B).
Four mutants of S. Typhimurium, with Tet-conditional growth, were created by inserting the tetRA cassette.
For this vector, we also inserted the Gateway Destination cassette after the WPRE (Figure 3E).
