T7 Riboregulation System: Experimental Design

Strains, Plasmids, and Reagents
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.

Anti-Fouling Peptide Construct: Experimental Design

We hypothesize that we can develop an improved version of the current adhesives by developing a fusion protein of Mgfp-5 with Mefp-1 as the anchoring region for the anti-biofouling peptide. An integral part of developing this peptide is to co-translationally insert L-DOPA into our peptide, which has never been done before with mussel foot proteins (Figure 5). In this process of orthogonal translation, we first will get rid of the UAG stop codon and then transform the strain to synthesize tRNA and tRNA transferase that corresponds to the UAG codon and the L-DOPA non-standard amino acid to develop the GRO. The advantage of this procedure is that we have the ability to skip the time-consuming and inefficient tyrosinase enzyme treatment step.

Protein Purification We plan to purify the protein by using the Twin Strep Tag in tandem with the Flag tag, which was included in out master construct of the anti-biofouling peptide (Figure 6). The Flag tag is perfectly cleavable by the enzyme enterokinase. The FLAG tag is made up of 8 amino acids and works well for low-abundance proteins. It is hydrophilic, so it will most likely not interfere with protein folding and function of the target protein. The Strep tag is also made up of 8 amino acids that will not disturb the protein’s functions. We chose the FLAG tag because it is perfectly cleavable. Info on LL-37 and N-terminus? The protein will be purified in a Strep-Tactin® Sepharose® column. In order to address the L-DOPA adhesive L-DOPA component, our final step is to elute with a base to reduce the amount of the anti-biofouling peptide that sticks to the column due to L-DOPA adhesion (Figure 7).
Flag Tag Sequence: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
Strep Tag Sequence: Sequence: Trp-Ser-His-Pro-Gln-Phe-Glu-Lys

Characterization of Coating Adhesion Properties

We hypothesize that we can develop an improved version of the current adhesives by developing a fusion protein of Mgfp-5 with Mefp-1 as the anchoring region for the anti-biofouling peptide. An integral part of developing this peptide is to co-translationally insert L-DOPA into our peptide, which has never been done before with mussel foot proteins (Figure 5). In this process of orthogonal translation, we first will get rid of the UAG stop codon and then transform the strain to synthesize tRNA and tRNA transferase that corresponds to the UAG codon and the L-DOPA non-standard amino acid to develop the GRO. The advantage of this procedure is that we have the ability to skip the time-consuming and inefficient tyrosinase enzyme treatment step.

Protein Purification We plan to purify the protein by using the Twin Strep Tag in tandem with the Flag tag, which was included in out master construct of the anti-biofouling peptide (Figure 6). The Flag tag is perfectly cleavable by the enzyme enterokinase. The FLAG tag is made up of 8 amino acids and works well for low-abundance proteins. It is hydrophilic, so it will most likely not interfere with protein folding and function of the target protein. The Strep tag is also made up of 8 amino acids that will not disturb the protein’s functions. We chose the FLAG tag because it is perfectly cleavable. Info on LL-37 and N-terminus? The protein will be purified in a Strep-Tactin® Sepharose® column. In order to address the L-DOPA adhesive L-DOPA component, our final step is to elute with a base to reduce the amount of the anti-biofouling peptide that sticks to the column due to L-DOPA adhesion (Figure 7).
Flag Tag Sequence: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
Strep Tag Sequence: Sequence: Trp-Ser-His-Pro-Gln-Phe-Glu-Lys