One percent agarose gels were made with 0.5% TBE obtained from American Bio and stained with either ethidium bromide (Sigma-Aldrich) in the case of screening or SYBR Safe (Invitrogen) in the case of cloning. Gel extraction and purification was completed with QIAprep Gel Extraction Kit following the protocol provided. PCR purification was accomplished with the QIAquick PCR Purification Kit, following the protocol provided. Plasmid purification was accomplished using the QIAprep Spin Miniprep Kit and the protocol provided. For all DNA kits provided by QIAgen we used Denville Spin Columns for Nucleic Acid Purification. The concentration of DNA was measured using a Biotek Synergy HT Multi-Mode microplate Reader with accompanying Take3 Microvolume plates. All restriction enzymes, and Gibson Assembly Master Mix are from New England Biolabs. Hifi HotStart Readymix and 2GFAST Readymix with loading dye for PCR were obtained from KAPA Biosystems.

A second promoter, P_LtetO, which is induced by ATC, will express the taRNA capable of interacting with the crRNA and releasing the RBS for docking of the T7 RNA polymerase. This will expose the RBS and allow for translation of T7 RNA Polymerase. Once the T7 RNA Polymerase is expressed, it can then bind to the T7 Promoter and lead to the expression of the gene of interest, such as LL-37. The ribo-regulated T7 RNA Polymerase (formally known as α12c) and the TolC selection marker will be ultimately incorporated into a conjugative plasmid and into the genome of E. coli to control for copy number. In this way, the cell can better regulate protein expression. A second pZ plasmid will contain the gene of interest expressed by a T7 promoter. Finally, the third plasmid will contain the OTS.

The benefit of this type of system is that it is robust and can be easily re-engineered, portable in the form of plasmids, compatible across multiple E. coli strains, and efficient in that it does not require the cell to expend more energy on the constitutive synthesis of another protein. We hypothesize that by utilizing these two levels of control, we will be able to reduce the expression of T7 RNA polymerase and produce a system with zero basal expression of the gene of interest.