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The transcription ratios of the pfkA gene of the three transformants that gave the highest itaconic acid productivity at time point 30 h (T3, T5 and T7) were compared with the highest itaconic acid producing strain that expresses the cluster genes cadA, mttA and mfsA (C3) and with the parent strain CAD.
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PCR products that gave high levels of transformants on quadruple dropout media were analyzed further by PCR amplification of three different clones to confirm single insert size and by DNA sequencing.
Within each subgroup, the condition that gave the largest number of transformants was set to "+ + + + +"; there is a "-" when no transformants were recovered.
Using this procedure, it is possible to get cells that give 10-10 transformants per mg of plasmid, when 10 ng are used for transformation.
Transformants that were generated through co-transformation with plasmid pHsp70A-GLuc were also analyzed qualitatively for luciferase activity, and several of these transformants showed chemiluminescence (data not shown).
Transformants that were generated through co-transformation with plasmid pPsaD-GLuc were analyzed for luciferase activity.
Given the number of promoter variants (256), the number of possible polymerase variants (8,000), and the number of transformants that were assessed in each round (10 – 10), it is likely that there was a population bottleneck during the first round of selection.
The transformants that were able to grow on resistant plates were considered genetically stable.
Leu+ transformants were replica-plated onto YPD plates at 36°C, and the plasmid DNA was recovered from transformants that exhibited plasmid-dependent rescue.
We obtained Chloramphenicol-resistant R. montanensis transformants that expressed GFPuv from a single chromosomally integrated transposon, but they were substantially less fluorescent than the R. monacensis GFPuv transformant.
I64 T0 primary transformants that phenotypically resembled cv.
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