The product concentration was improved 8.6-fold compared with E. coli BL21(DE3)/pET-SUMO_VvGT14ao yielded the highest titres. coli expression strains, resulting in 18 strains that were tested for glycosylation efficiency with terpenols and a phenol. Seven expression plasmids differing in the resistance gene, origin of replication, promoter sequence, and fusion protein tag were generated and transformed into four different E. coli expressing VvGT14ao, a glycosyltransferase gene from grape ( Vitis vinifera). The aim of this work was to increase the titre of terpenyl glucosides in biotransformation assays with E. However, there is still a need to optimize the biocatalysts. Total time does not include transformation, isolation or analysis.Glycosides are becoming increasingly more relevant for various industries as low-cost whole-cell-biocatalysts are now available for the manufacture of glycosides. If the source for gene transfer is gDNA, add 2 hours to calculation for the traditional cloning method. Ligation Independent Cloning (LIC) Workflow Note that times are based on estimates for moving a gene from one plasmid to another. Some types of sequence modifications not possible.This method allows rapid creation of compatible ends and avoids the clean-up steps prior to vector and insert joining common to many successful cloning methods. One of these interesting approaches is the use of nicking DNA endonucleases (NiDE) to create the complementary overlaps used to anneal the vector and insert. Other variations on the basic LIC design principles have been published and put into practice in various labs. This modification of the protocol allows a scarless and sequence-independent insertion into most any vector to be made. The product contains 4 nicks, like the original LIC product, and is repaired by E.coli during transformation. After the overlap is generated, dCTP is added back to the reaction, shifting the enzyme back into a polymerase, where it stalls due to the lack of a complete set of dNTPs in the buffer. This allows the exonuclease activity of T4 DNA Polymerase to proceed and generate the complementary overlaps between insert and vector. In this variation, all dNTPs are initially excluded from the reaction. One in particular, sequence and ligation independent cloning (SLIC), has been adopted by many researchers. More recently, the technique has evolved to include many useful variations. This technique allows efficient creation of scarless recombinant plasmids at many, but not all, positions in a vector. Joined fragments have 4 nicks that are repaired by E.coli during transformation. Incorporation of dGTP in the reaction limits the exonuclease processing to the first complementary C residue, and not present in the designed overlap, where the polymerization and exonuclease activities of T4 DNA Polymerase become “balanced”. This creative technique uses the 3’ → 5’ exo activity of T4 DNA Polymerase to create overhangs with complementarity between the vector and insert. Inserts are usually PCR amplified and vectors are made linear either by restriction enzyme digestion or by PCR. Ligation Independent Cloning (LIC) is a technique developed in the early 1990s as an alternative to restriction enzyme/ligase cloning.
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