Stable Maintenance of Multiple Plasmids in E. coli Using a Single Selective Marker

Plasmid-based genetic systems in Escherichia coli are a staple of synthetic biology. However, the use of plasmids imposes limitations on the size of synthetic gene circuits and the ease with which they can be placed into bacterial hosts. For instance, unique selective markers must be used for each plasmid to ensure their maintenance in the host. These selective markers are most often genes encoding resistance to antibiotics such as ampicillin or kanamycin. However, the simultaneous use of multiple antibiotics to retain different plasmids can place undue stress on the host and increase the cost of growth media. To address this problem, we have developed a method for stably transforming three different plasmids in E. coli using a single antibiotic selective marker. To do this, we first examined two different systems with which two plasmids may be maintained. These systems make use of either T7 RNA polymerase-specific regulation of the resistance gene or split antibiotic resistance enzymes encoded on separate plasmids. Finally, we combined the two methods to create a system with which three plasmids can be transformed and stably maintained using a single selective marker. This work shows that large-scale plasmid-based synthetic gene circuits need not be limited by the use of multiple antibiotic resistance genes

Structure-Based Stabilization of HIV-1 gp120 Enhances Humoral Immune Responses to the Induced Co-Receptor Binding Site

The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein, gp120, possesses conserved binding sites for interaction with the primary virus receptor, CD4, and also for the co-receptor, generally CCR5. Although gp120 is a major target for virus-specific neutralizing antibodies, the gp120 variable elements and its malleable nature contribute to evasion of effective host-neutralizing antibodies. To understand the conformational character and immunogenicity of the gp120 receptor binding sites as potential vaccine targets, we introduced structure-based modifications to stabilize gp120 core proteins (deleted of the gp120 major variable regions) into the conformation recognized by both receptors. Thermodynamic analysis of the re-engineered core with selected ligands revealed significant stabilization of the receptor-binding regions. Stabilization of the co-receptor-binding region was associated with a marked increase in on-rate of ligand binding to this site as determined by surface plasmon resonance. Rabbit immunization studies showed that the conformational stabilization of core proteins, along with increased ligand affinity, was associated with strikingly enhanced humoral immune responses against the co-receptor-binding site. These results demonstrate that structure-based approaches can be exploited to stabilize a conformational site in a large functional protein to enhance immunogenic responses specific for that region

Examination of the C-terminal assembly motif in lactose repressor

Assembly requirements for lactose repressor protein have been examined by substitutions in the leucine heptad repeat (LHR) tetramerization domain. All a and d positions of the LHR were substituted with Ile (II) or a positions with Leu and d positions with Ile (ILI). Additionally, identical substitutions made in the previously-constructed LacI/GCN4 LHR chimera, Izip were termed IzI and IzLI. To express these proteins, a system was designed employing the popular T7&phis; promoter without usual repression by lactose repressor protein. Cloning T7 gene 1 under arabinose repressor control on pBAD33 (Guzman et al., 1995) generated the plasmid pTara. Following co-transformation of pTara and expression plasmid, production of both T7 RNA polymerase and target protein was induced with L-arabinose. High expression levels of lactose repressor mutant proteins were obtained, and the general applicability of this system was confirmed using human p53 and Drosophila Ubx proteins. Proteins II and ILI demonstrated elution profiles from column chromatography typical of predominantly monomeric assembly, whereas IzI and IzLI eluted at comparable ionic strengths to wild type LacI. Gel filtration chromatography indicated that IzI is of the correct size for hexameric protein, and IzLI is in an equilibrium between tetramer and monomer. IzI and IzLI bound neither wild type operator nor completely palindromic operator sequence with specific affinity, and no decrease in operator binding was observed for either protein with inducer. IzI and IZLI bound inducer with wild type affinity, but did not exhibit the wild type reduction in affinity normally observed at pH 9.2. Trypsin digestion studies indicated that both of these proteins were reduced to lower molecular weight bands than wild type lactose repressor and that IzLI is digested to these lower weight states more rapidly than IzI. Urea denaturation studies yielded midpoints of denaturation for both proteins lower than wild type lactose repressor protein, and less steep slopes for the unfolding/dissociation transitions. These data are collectively consistent with folding to a wild type monomeric tertiary structure but anomalous assembly to dimer and higher order oligomers. The changes in dimer assembly apparently have consequences for operator recognition and cooperativity of inducer binding

Transcriptional Activity of HTLV-I Tax Influences the Expression of Marker Genes Associated with Cellular Transformation

Human T cell leukemia virus type I (HTLV-I) has been identified as the etiologic agent of adult T cell leukemia (ATL). HTLV-I encodes a transcriptional regulatory protein, Tax, which also functions as the viral transforming protein. Through interactions with a number of cellular transcription factors Tax can modulate cellular gene expression. Since the majority of Tax-responsive cellular genes are important regulators of cellular proliferation, the transactivating functions of Tax appear to be necessary for cellular transformation by HTLV-I. Gaining a complete understanding of the broad range of genes regulated by Tax, the temporal pattern of their expression, and their effects on cell function may identify early markers of disease progression mediated by this virus