The T7 Riboregulation System works by a “three-lock system.” The first lock is the cis- repressing RNA (crRNA), which is induced bysopropyl β-D-1-thiogalactopyranoside (IPTG). The second lock is the trans-activating RNA (taRNA), which is induced by anhydrous tetracycline (ATC). If the taRNA is unlocked, it will bind to the crRNA, removing the hairpin and making the ribosomal binding site accessible for ribosomal binding, leading to translation of a specific protein, in this case, T7 RNA Polymerase. This system was initially developed by Dr. Farren Isaacs, and has been shown to work with chloramphenicol resistance (chloramphenical acetyl transferase gene) in place of the T7 gene. The plasmid was synthesized via Gibson assembly, and confirmed by sequencing.

Currently the riboregulation system may have an issue with the internal T7 sequence, and while sequencing has been done, no successful data has been obtained.

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 after subjecting the substrate to erosion. 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.