Saturated overnight cultures of E. coli carrying the chromosomal gam-gfp expression cassette were diluted 1 100 in fresh medium, 100 ng/ml doxycycline added to induce GamGFP, incubated for 1 hr shaking at 37°C, then 0.1% arabinose added to induce I- SceI production and incubated 3 hr more, then subjected to microscopic examination.
Gaming became about personal expression and that's incredible.
We investigated the potency of gene activated matrix (GAM) and implemented the GAM strategy in order to achieve a control of gene expression, as well as a specific cell recruitment.
gam sog pattern similar to expression from other known An. gam sog CRMs.
We constructed a regulatable chromosomal expression cassette of Mu gam and a Mu gam-gfp fusion gene in the E. coli chromosome, controlled by the doxycyline/tetracycline-inducible PN25 tetO promoter ('Materials and methods', Figure 1A).
This pattern is consistent with the described expression of An. gam sog, which is mesodermally, not ectodermally, expressed, perhaps due to utilization of a different combination of cis-regulatory inputs, or of the same inputs with different strengths, by the responsible CRM (Goltsev et al. 2007).
For experiments assaying GamGFP co-localization with TetR-mCherry on a chromosomal tetO array, we required and constructed a TetR-independent expression cassette for gam-gfp.
We call these coordination GAM, control GAM, and separate GAM.
gam sog CRM drives reporter gene expression in the mesoderm (An. gam_sog_1; fig. 5 a. I).
Expression of γ-toxin (gam) under control of the RP40 promoter resulted in auxin-related defects, resembling those of elp mutants (13/20 lines analyzed).
