pMnt/Lac is constitutively on but is repressed by the LacI protein. The promoter is also repressed by the Mnt protein. The addition of IPTG should repress the LacI repressor and glow in the absence of the Mnt protein.
To Test repression of promoter
Some promoter→RBS+LacI+t+t+pLac/Mnt→RBS+GFP ''does not glow''
Some promoter→RBS+Mnt+t+t+pLac/Mnt→GFP ''does not glow''
Some promoter→RBS+LacI+RBS+Mnt+t+t+pLac/Mnt→GFP ''does not glow''
To Test the promoter and addition of IPTG
Some promoter→IPTG+RBS+LacI+t+t+pLac/Mnt→GFP ''glows''
Some promoter→IPTG+RBS+LacI+Mnt+t+t+pLac/Mnt→GFP '' glows''
|LacI||Mnt||IPTG + GFP||IPTG - GFP|
Steps to Build Hybrid Promoter
Perform a Ligation of Mnt/Lac (K091104) with E0240 (RBS + GFP)
Transform ligation over night
Clones were picked and set over night in LB + AMP