E. coli strains used in this study included BL21(E. coli B F- dcm ompT hsdS(rB- mB-) gal [malB+]K-12(λS)), BL21(DE3)( F– ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])), ECNR2(ΔmutS:cat.Δ(ybhB-bioAB): [λcI857.Δ(cro-ea59):tetR-bla]), Mach1(ΔrecA1398 endA1 tonA Φ80ΔlacM15 ΔlacX74 hsdR(rK- mK+)), and 730. Strains used for transformation were grown in LB min (Cold Spring Harbor Protocols 2006). Cells used for cloning and mini-prep were grown in selective medium of 2XYT (2xYt Medium (7281) 2010) with either kanamycin (American Bioanalytical) or spectinomycin (Sigma-Aldrich). Kanamycin and streptomycin were used at 30 mg/mL and 95 mg/mL respectively.

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.

The P_LlacO promoter controls the expression of the crRNA and is induced by IPTG. As specified above, we will use artificial riboregulatory elements to restrict translation of the mRNA sequence encoding the T7 RNA Polymerase. Specifically, the crRNA sequence will be inserted downstream of the promoter driving T7 RNA Polymerase and upstream of the ribosomal binding site (RBS).

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.

Preliminary proof of concept testing was conducted on a commercially available MAP-based product known as Cell-Tak ^TM. Cell-Tak^TM is designed to facilitate cell adhesion to normally non-biocompatible surfaces such as microscope slides and petri dishes. We deposited ~20 µg films of Cell-Tak onto borosilicate substrates and proceeded to erode them under deionized H₂O and 5% acetic acid. The results from this experiment are presented below and illustrate the design of our assay to test a variety of solvent and erosion conditions on MAP films. A balance that can read to uncertainties of 1 µg was used to determine the mass of protein remaining. An exponential decay curve was fitted to these experiments giving decay rates of 0.002 µg/pass and 0.046 µg/pass for deionized H₂O and 5% acetic acid, respectively. As lower pH reverses the coordination of L-DOPA, it is expected that the acidic conditions engender the higher rate of decay. This experiment presents a preliminary result that validates our ability to apply erosion onto MAP-coated surfaces. We intend to apply a similar protocol to metal and plastic surfaces as well as erode surfaces under different pH conditions to provide a more comprehensive picture of the optimal conditions for mussel adhesion.

A contact angle measurement of a Cell-Tak^TM was recorded and served as an indication for presence of peptide on surfaces. The contact angle is measured between the surface of the drop and the table-top. Larger contact angles are indicative of more hydrophobic surfaces while shallower contact angles correspond to more wettable surfaces. A contact angle of 25.053º was obtained between an untreated silica substrate and a 2 µL drop of DI H₂O. However, when surfaces were treated with the MAP, the contact angle increased to 62.007º, indicative of an increase in the hydrophobicity of our substrate. This result validates the evolutionary need for mussels to secrete proteins that are resistant to water in order to survive and anchor themselves in constantly wet environments.