The inverse PCR was performed with primers facing outwards towards the ends of gcu14, bla GES-1 and aacA4 so that only circular gene cassette configurations would be amplified.
In an attempt to optimise production of cellulolytic enzymes in transplastomic tobacco, we used a set of endogenous and heterologous cis-acting elements to construct several cassettes with varying configuration of the cis-acting regulatory elements and the foreign genes expressed.
In cases where the three cassettes are identified, their configuration is apparently similar to C. glabrata's: both HML- and MAT-like cassettes are on the same scaffold, while the HMR-like cassette is on a different one, except for N. delphensis, where the three cassettes are on the same scaffold.
Structural design (configuration) of a cassette can vary according to the plastome locus where integration is planned; usually transcriptionally-active or silent intergenic spacer as well as cassettes of different configuration can be introduced into the same plastome locus.
The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW), had a higher transcription level than bla GES-1/ aacA4, indicating that the cassette position affects the transcript amounts.
We hypothesized that testing levels of accumulation of the foreign proteins produced from different cassettes would determine an ideal configuration, capable of expressing other recombinant proteins at high levels.
gcu14, the first cassette in integrons with the weak PcW configuration, presented approximately two-fold higher transcription when compared to bla GES-1 and aacA4 separately or when the fused cassette bla GES-1/ aacA4 was considered.
Many chloroplast transformation vectors with different configuration of the chloroplast expression cassettes were designed and applied [ 7, 11, 16, 17].
The configuration and manufacturing design of the diagnostic cassette and the installation of optical diagnostic systems within the cassette are described.
