RESUMO
Traditionally engineered genetic circuits have almost exclusively used naturally occurring transcriptional repressors. Recently, non-natural transcription factors (repressors) have been engineered and employed in synthetic biology with great success. However, transcriptional anti-repressors have largely been absent with regard to the regulation of genes in engineered genetic circuits. Here, we present a workflow for engineering systems of non-natural anti-repressors. In this study, we create 41 inducible anti-repressors. This collection of transcription factors respond to two distinct ligands, fructose (anti-FruR) or D-ribose (anti-RbsR); and were complemented by 14 additional engineered anti-repressors that respond to the ligand isopropyl ß-d-1-thiogalactopyranoside (anti-LacI). In turn, we use this collection of anti-repressors and complementary genetic architectures to confer logical control over gene expression. Here, we achieved all NOT oriented logical controls (i.e., NOT, NOR, NAND, and XNOR). The engineered transcription factors and corresponding series, parallel, and series-parallel genetic architectures represent a nascent anti-repressor based transcriptional programming structure.
Assuntos
Bioengenharia/métodos , Repressores Lac/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Expressão Gênica/fisiologia , Redes Reguladoras de Genes , Repressores Lac/síntese química , Ligantes , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/síntese química , Biologia Sintética/métodos , Fatores de Transcrição/síntese química , Fatores de Transcrição/metabolismoRESUMO
Lactose repressor protein (LacI), a negative transcriptional regulator in Escherichia coli, relies on an allosteric conformational change for its function. The LacI effector isopropyl-ß,D-thiogalactoside (IPTG) promotes this allosteric response and engages the side chains of residues N125 and D149 based on the crystallographic structure of LacI·IPTG. Targeted molecular dynamics (TMD) simulations have indicated involvement of these side chains during the protein structural changes in response to inducer binding. To examine this region further, we applied stochastic boundary molecular dynamics (SBMD) simulation and identified a transient interaction between residues N125 and D149. On the basis of these data, we introduced substitutions for either/both residues and analyzed their impact on protein function. The substitutions utilized were alanine to preclude hydrogen bonding or cysteine to allow disulfide bond formation, which was not observed for N125C/D149C. Minimal impacts were observed on operator affinity for all substitutions, but D149C, N125A/D149A, and N125C/D149C bound to IPTG with 5-8-fold lower affinity than wild-type LacI, and exhibited decreased allosteric amplitude (K(RI/O)/K(R/O)). Of interest, the double mutants did not exhibit an allosteric response to an alternate inducer, 2-phenylethyl-ß,D-galactoside (PhEG), despite demonstration of PhEG binding. Further, the presence of the anti-inducer, o-nitrophenyl-ß,D-fucoside (ONPF), enhanced operator affinity for wild-type LacI and all other mutant proteins examined, but behaved as an inducer for N125A/D149A, decreasing operator binding affinity. These results confirm the role of residues 125 and 149 in ligand binding and allosteric response and illustrate how readily the function of a regulatory protein can be altered.
Assuntos
Substituição de Aminoácidos/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Repressores Lac/antagonistas & inibidores , Repressores Lac/química , Lactose/química , Regulação Alostérica/genética , Asparagina/genética , Ácido Aspártico/genética , DNA Bacteriano/antagonistas & inibidores , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Glicosídeos/química , Glicosídeos/genética , Óperon Lac/genética , Repressores Lac/genética , Lactose/genética , Ligantes , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Conformação Proteica , Multimerização Proteica/genéticaRESUMO
A simple strategy was tested for imposing artificial regulation of rhizobial genes during nodule development. Isopropyl-ß-d-1-thiogalactoside (IPTG) was added to liquid root media to sustain expression of rhizobial genes controlled by Escherichia coli lac promoter/operators and repressor gene lacI. Conversely, a rinsing protocol was devised to remove IPTG sufficiently that genes could be repressed after having been induced. gusA under this control exhibited clearly delineated expression and repression in both the determinate Rhizobium etli-Phaseolus vulgaris and the indeterminate Sinorhizobium meliloti-Medicago sativa symbioses. Apparently, IPTG was taken up in sufficiently undegraded concentrations that gene expression was derepressed even in interior portions of the nodule. Moreover, the rinsing protocol led to obvious repression of gusA. Importantly, no deleterious effects of IPTG on nodule development, infection, or nitrogen fixation were observed. An R. etli CE3 gene required for lipopolysaccharide O antigen and infection on bean was put under this control by means of a two-plasmid construct. When this construct was added to a strain with a null mutation in this gene, infection, nodule development, and nitrogenase activity all depended on the length of time before IPTG was rinsed from the roots after inoculation.
