Methods Overview

Different Methods will be presented below.

Strains and Media

scherichia coli Top10 (Invitrogen) was used for all transformations and assays. Media included SOB for transformations and LB for overnight cultures. Transformed strains were grown at 37°C using Ampicillin resistance. Primers for PCR were purchased from Integrated DNA Technologies (IDT) and New England Biolabs (NEB) donated all of the restriction enzymes. All sequencing was conducted by the Northwestern Genomics Core.

Forming a Library of Constructs

e low copy plasmid pSB4A5 will be used for the different promoter constructs. The asr and gadA promoters were extracted from the E. coli genome via colony polymerase chain reaction (PCR). The constitutive promoters TacI and Lpp were amplified from pDAK1 and pDAK2 donated by the Jewett Lab6,7. The restriction enzyme cut sites EcoR1, Pst1, Spe1, and Xba1 were used in ligation to create the different constructs (Figure 3). When multiple parts were connected a mixed site was formed between Spe1 and Xba1, which cannot be cut, by any of the restriction enzymes. This leads to the benefit of not having a restriction site in the middle of the construct, and furthermore these standard restriction enzymes can always be used with the final dual-state promoter constructs.