T7 Riboregulation System: Experimental Design

• Adhesion:
  • Subject peptide coated surfaces to liquid erosion:
    • A number of ASTM assays used in industrial coating testing were investigated, but none offered the level of quantitation desired for our applications. Therefore, an original rig was designed and built to introduce liquid based erosion by laminar flow through a bath. This system directly mimics the drag that a coated surface might experience on a ship's hull. Precise specifications of the rig are provided in a separate section below.
    
    
    Figure X. A diagram illustrating the configuration of the erosion rig developed to introduce coated surfaces to liquid erosion.
    
    
  • Assay presence of peptide on eroded surfaces:
    • Quartz Crystal Microbalance (QCM): A QCM is capable of measuring mass per unit area on a very sensitive scale. The QCM used in these experiments recorded masses with ±1 ng/cm² uncertainty. The way this instrument works is by measuring change in the resonance frequency, which is converted into a mass estimate on the basis that resonance frequency will decrease with increasing mass. We intend to subject the quartz crystals to varying levels of erosion and determining coating retention from the QCM mass measurement. Alternatively, various flow cell and in-situ erosion techniques can be coupled to the QCM to show the real-time changes in resonance frequency due to loss of mass. Several labs and facilities assisted with planning and execution of this measurement, including Dr. Michael Rooks at the Yale Institute for Nanoscience and Quantum Engineering and Dr. Islam Mosa in the lab of Dr. James Rusling in the Department of Chemistry at the University of Connecticut.
    • Total Protein Staining and Fluorescence Imaging: Coomassie Blue was used as a total protein stain to determine presence of coating on eroded slides. Adsorbed protein content can theoretically be determined visually from density of stain. Since our construct was designed with an sfGFP domain, we intend to assay presence of our peptide with fluorescence.
    • Contact Angle Measurement: A contact angle measurement of protein coated silica substrates was conducted as an indicator for presence of peptide, protein hydrophilicity/hydrophobicity, and surface energy. Wetting surfaces show a shallow contact angle, while hydrophobic surfaces show a larger contact angle. A contact angle characterizes the wettability of a surface and Young's equation can be used to determine interfacial energies between the three phases in equilibrium, given below. Note that γ_XY corresponds to the interfacial energy between phase X and phase Y.
      0=γ_SG – γ_SL – γ_LSCos(θ_C)
      This measurement was conducted with the assistance of Dr. Raphael Sarfati, Dr. Katharine Jensen, and Dr. Rostislav Boltyanskiy in the lab of Dr. Eric Dufresne in the Yale Department of Mechanical Engineering.
    • Fourier Transform Infrared Spectroscopy (FTIR): As a further test to determine if material is adhered to surfaces, we will use Fourier Transform Infrared Spectroscopy (FTIR). The cured adhesive film should exhibit a different spectrum than the uncured adhesive. A notable difference would speak to a change in vibrational bond energies caused by coordination or bonding to our surface.
  • Assay peptide adhesion strength:
    • Atomic Force Microscopy (AFM): The standard for measurement of the force of adhesion of MAPs is AFM. This type of measurement is known as a "pull-off" force determination and involves depressing an AFM cantilever functionalized with a 20 µm bead until it comes in contact with a coated substrate surface. The instrument then determines the force required to remove the cantilever from the substrate.
      
      
      Figure X. This is an AFM cantilever with a 20 µm silica bead fixed to the tip. By functionalizing the tip, we can control the adhesion interface for which we test our MAP adhesives. In this case, we intend to use a silica bead to measure the adhesion of our coating to a silica interface. This measurement was conducted with the assistance of Dr. Michael Rooks at the Yale Institute for Nanoscience and Quantum Engineering.

      
      

    • Optical Tweezers: While AFM has been used in many MAP studies successfully to measure MAP adhesion force, it comes with some limitations. Inevitably, there is some significant contact area, which makes the adhesion measurement read the adhesive force of multiple proteins. However, the technology exists to measure adhesion on the individual protein level. Some studies have measured the adhesion force of L-DOPA on the single molecule level by chemically linking the L-DOPA residue to the AFM cantilever. However, no such study have looked at adhesion force on the single protein level. Using high intensity lasers, one can engender a repulsive force between two beads in relation to their refractive indices. We intend to link our MAP to a biotin functionalized bead and measure its adhesion to a silica bead substrate.
      
      Figure X. This diagram illustrates how a DNA handle can be linked to a protein of interest to bind the protein to an optical tweezer bead into which the high intensity laser can be fired to engender a pull force. We intend to conduct a similar protocol with our adhesive peptide. ⁸ This measurement was conducted with the assistance of Dr. Junyi Jiao in the lab of Dr. Yongli Zhang from the Yale Department of Cell Biology.

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.
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.

Two Levels of Regulation for T7 Polymerase Expression
Our goal is to reduce the expression of T7 RNA polymerase and create an efficient system for the expression of heterologous proteins in E. coli. In order to carry this out, we chose to introduce two levels of regulation. The first level of regulation will be at the transcriptional level. We will use pZE21 of the pZ system of vectors developed by Lutz and Buschard with the PLlacO promoter to inhibit the expression of T7 RNA polymerase. The PLlacO promoter controls the expression of the crRNA (cis repressing RNA) and is induced by IPTG (isopropyl-beta-D-thiogalactopyranoside). The second level of regulation will occur at the translational level. We will use the artificial riboregulatory elements (Figure 2) devised by Isaacs et al. to restrict translation of the mRNA sequence encoding the T7 RNA Polymerase (Isaacs et al., 2004). The cis-repressing RNA (crRNA) sequence will be inserted downstream of the promoter driving T7 RNA Polymerase and upstream of the ribosomal binding site (RBS). The crRNA is complimentary to the RBS and forms a stem loop at the 5’ end of the mRNA segment, blocking ribosomal docking and translation. A second promoter, PLtetO, which is induced by ATC, will express the trans-activating RNA (taRNA) capable of undergoing a linear-loop interaction competitively interacting with the crRNA and releasing the RBS for docking of the T7 RNA polymerase . that 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 the antimicrobial peptide (Figure 3).
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 (Figure 2a and 2b). The reason why it is important to control for copy number is that the copy number of the pZE21 backbone is fairly large. This means that it will be more challenging for the cell to regulate protein expression, so a low copy number would enable better cell regulation of proteins. A second pZ plasmid will contain the gene of interest expressed by a T7 promoter. Finally, the third plasmid will contain the orthogonal translation system (Figure 3a and 3b). 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.

Anti-Fouling Peptide Construct

Construct Synthesis and Expression: Strains, Plasmids,
The construct sequence was synthesized by Genscript and shipped as pUC57-Kan_2StrepFLAGLLFP151GFP, and transplanted to the pZE21 plasmid, pZE21_2StrepFLAGLLFP151GFP (BBa_K1396000).
All plasmids were first grown in Mach1, then purified and retransformed into C31POE.ompT.lon.endA.ΔtolC, a recoded strain with all amber stop codons (TAG) replaced, and Release Factor 1 replaced with Streptomycin resistance. It is thus able to encode nonstandard amino acids such as L-DOPA, the incorporation of which is facilitated by the DOPA orthogonal translation system (OTS).
A second strain was made with the Tyrosine suppressor system transformed instead, so the construct can be expressed with tyrosines in the place of L-DOPA, as L-DOPA is very toxic to cells. The construct was then separated into smaller constructs such as pZE21_LLFP151 (BBa_K1396001), pZE21_FP151GFP (BBa_K1396002), pZE21_FP151 (BBa_K1396003).

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 genetically recoded organism (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