Conversion of LuxR：转换的LuxR.pptVIP

Conversion of LuxR How? What is LuxR / lux? In this experiment… Results (Fig. 2)! Found that with the experimental plasmid, b-galactosidase production stopped when 3-oxo-C6-HSL was added (this usually activated LuxR, but it appears to be repressed) With control lacZ plasmid, b-galactosidase production occurred with or without 3-oxo-C6-HSL Results (Fig. 3)!! The higher the concentration of 3-oxo-C6-HSL, the greater the repression by the lux box plasmid. The higher the concentration of a similar compound, C8-HSL, the greater the repression This is the OPPOSITE of what would happen normally Results (Fig. 4)!! LuxR represses lacZ even when it’s downstream of the promoter Results (Fig. 5)!! Adding a lot of lux boxes elsewhere made repression of lacZ less efficient, showing that repression is actually caused by LuxR binding to the lux boxes Results (Fig. 6)!! Analysis of mutant LuxR’s LuxR has an N-terminus that either binds to the signal OR binds to the C-terminus LuxR has a C-terminus which is bound to RNAP and lux box OR the N-terminus Results (Fig. 6)!! C-terminal deletions resulted in an inability to bind to RNAP Normally, this lessens the transcription level In this experiment, it increases transcription level (lowers repression level) N-terminal deletions of 5 or 10 AA’s resulted in signal-dependent repressors N-terminal deletions of 20 and 127 AA’s resulted in loss of repression ability Results (Table 2)!! Which came first, the chicken or the egg? Rephrased: Which came first, signal binding or DNA binding? Hopefully obvious to synthetic biologists, signal binding comes first Looked at single AA substitutions in LuxR to determine this Conclusions LuxR acts as a repressor when the lux box is in between the RNAP binding consensus sequences LuxR needs both its N-terminal and C-terminal ends to function as a repressor * LuxR Transcription Some Scientific Tinkering… LuxR Transcription bp -42.5 0 -35 -10 Transcriptional Start Site RNA Polymerase Binding Site Consensus S