Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells, yet cell-free approaches offer simpler and more flexible contexts. Here, we evaluate cell-free regulatory systems based on T7 promoter-driven expression by characterizing variants of TetR and LacI repressible T7 promoters in a cell-free context and examining sequence elements that determine expression efficiency. Using the resulting constructs, we then explore different approaches for composing regulatory systems, leading to the implementation of inducible negative feedback in Escherichia coli extracts and in the minimal PURE system, which consists of purified proteins necessary for transcription and translation. Despite the fact that negative feedback motifs are common and essential to many natural and engineered systems, this simple building block has not previously been implemented in a cell-free context. As a final step, we then demonstrate that the feedback systems developed using our cell-free approach can be implemented in live E. coli as well, illustrating the potential for using cell-free expression to fast track the development of live cell systems in synthetic biology. Our quantitative cell-free component characterizations and demonstration of negative feedback embody important steps on the path to harnessing biological function in a bottom-up fashion.

We screened a chicken liver cDNA expression library with a probe spanning the distal region of the chicken vitellogenin II (VTGH) gene promoter and isolated clones for a transcription factor that we have named VBP (for vitellogenin gene-binding protein). VBP binds to one of the most important positive elements in the VTGH promoter and appears to play a pivotal role in the estrogen-dependent regulation of this gene. The protein sequence of VBP was deduced from a nearly full length cDNA copy and was found to contain a basic/zipper (bZIP) motif. As expected for a bZIP factor, VBP binds to its target DNA site as a dimer. Moreover, VBP is a stable dimer free in solution. A data base search revealed that VBP is related to rat DBP. However, despite the fact that the basic/hinge regions of VBP and DBP differ at only three amino acid positions, the DBP binding site in the rat albumin promoter is a relatively poor binding site for VBP. Thus, the optimal binding sites for VBP and DBP may be distinct. Similarities between the VBP and DBP leucine zippers are largely confined to only four of the seven helical spokes. Nevertheless, these leucine zippers are functionally compatible and appear to define a novel subfamily. In contrast to the bZIP regions, other portions of VBP and DBP are markedly different, as are the expression profiles for these two genes. In particular, expression of the VBP gene commences early in liver ontogeny and is not subject to circadian control.Genetic and biochemical studies have established that transcription by RNA polymerase II requires the assembly of a stable preinitiation complex over the proximal promoter region of each target gene (6,32 which a nonfunctional partner suppresses the activity of a functional partner. In addition to DNA binding domains (and, in some cases, dimerization domains), transcription factors also contain one or more transactivation domains that are required to mediate positive effects on the general transcriptional machinery. Subclasses of transactivation domains have been identified, and it is likely that other novel domains will be found as more transcription factors are cloned and analyzed. Much current work is directed at understanding how these activation domains function, and the notion that additional bridging factors may be involved has been advanced from several recent studies (reviewed in reference 25).Studies in our laboratory are focused on a molecular understanding of the estrogen-dependent and liver-specific transcriptional regulation of the chicken vitellogenin II (VTGII) gene. The estrogen-dependent aspect of this regulation was shown to be due to the presence of two upstream estrogen response elements, and the ability of the VTGII promoter to be activated by these elements was found to be cell type specific (5, 7). A linker scanner mutational analysis of the VTGII promoter using transient expression assays in chicken hepatoma (LMH) cells (18) and chicken embryo fibroblast cells revealed that this promoter has multiple positive elements as well as a negat...

No abstract