DNA short sequences (operator sites) recognized by transcription factors have a key role for assembling regulated promoters. Three different sequences with high, medium and low binding affinity for the Lac repressor protein were synthesized. Single sequences were cloned as BioBricks (1) to allow their standard assembly with constitutive promoters. Thus, promoter transcriptional strength and repressor binding affinity can be independently fixed. Each of the three operators was cloned downstream a strong and a weak promoter. To develop a repressor generator the LacI gene was cloned downstream the weak promoter with the operator site, while the GFP reporter was cloned downstream the strong promoter with the same operator site. The generator was cloned on a medium copy number plasmid, while the reporter was cloned on a high copy number plasmid. Different promoter strength and plasmid copy number allowed the amplification of the signal. E. coli cells were transformed with both the plasmids to realize the closed loop configuration (test), and with only the reporter plasmid to have the open- loop configuration (control). Different fluorescence levels were observed in the three closed loop according to the repressor binding affinities. Closed loop constructs exhibited a lower fluorescence than the corresponding open.
Characterization of the Lac Repressor Operator Sites
CERONI, FRANCESCA;GIORDANO, EMANUELE DOMENICO;CAVALCANTI, SILVIO
2009
Abstract
DNA short sequences (operator sites) recognized by transcription factors have a key role for assembling regulated promoters. Three different sequences with high, medium and low binding affinity for the Lac repressor protein were synthesized. Single sequences were cloned as BioBricks (1) to allow their standard assembly with constitutive promoters. Thus, promoter transcriptional strength and repressor binding affinity can be independently fixed. Each of the three operators was cloned downstream a strong and a weak promoter. To develop a repressor generator the LacI gene was cloned downstream the weak promoter with the operator site, while the GFP reporter was cloned downstream the strong promoter with the same operator site. The generator was cloned on a medium copy number plasmid, while the reporter was cloned on a high copy number plasmid. Different promoter strength and plasmid copy number allowed the amplification of the signal. E. coli cells were transformed with both the plasmids to realize the closed loop configuration (test), and with only the reporter plasmid to have the open- loop configuration (control). Different fluorescence levels were observed in the three closed loop according to the repressor binding affinities. Closed loop constructs exhibited a lower fluorescence than the corresponding open.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
