<p>Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.</p>
+
+
<p>
+
Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.
+
</p>
+
+
</div>
+
+
<div class="column half_size">
+
<h5>What should this page contain?</h5>
+
<ul>
+
<li> Protocols </li>
+
<li> Experiments </li>
+
<li> Documentation of the development of your project </li>
The idea of cell free expression has existed for more than 20 years. It is the notion of taking the elements required for both transcription and translation and utilizing them for an external protein expression system. This is extremely beneficial for expression of cytotoxic proteins in which high yields are difficult to produce in vivo. For our purpose within this assay, this concept is utilized in conjunction with a regulatory factor for detection. This is detection is predicated either by regulation of the RBS at the transcript level or by sequestering the binding sight of RNA polymerase.
Work Flow
Identify trigger sequence and target plasmid or probe
qPCR utilizes the same basis as standard polymerase chain reaction, in which a target section of DNA can be identified and amplified via an enzymatic reaction (DNA Polymerase) and two ssDNA oligos (primers). Where qPCR differs is in the addition of either dye or probe based chemistry (this protocol will be primarily deal with the former). What this dye allows for is detection of newly formed DNA through the incorporation of a small fluorescent molecule into the new double stranded DNA (dsDNA). When the relative fluorescent units (RFU) being monitored rises above the background level (established by experimental controls) it is possible to ascertain a concentration of target gene. The relationship being the more concentrated the target the earlier in the thermocycler program signal will be generated.
Construct Synthesis and Expression: Strains, Plasmids, and Reagents
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 1). 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.
Figure 1. Integration of L-DOPA into peptide through orthogonal translation.
Protein Purification
We plan to purify the protein by using the Twin Strep Tag in tandem with the FLAG tag, which was included in our master construct of the anti-biofouling peptide (Figure 2).
Figure 2. A diagram illustrating the components in our final construct. The Twin Strep and FLAG tags are indicated.
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. The protein will be purified in a Strep-TactinR SepharoseR column. In order to address the 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 3).
Flag Tag Sequence: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
Strep Tag Sequence: Sequence: Trp-Ser-His-Pro-Gln-Phe-Glu-Lys
Figure 3. A diagram illustrating the proposed purification method.
Methods for Assaying Coating Adhesion Properties
Strains, Plasmids, and Reagents
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 4. 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 γXY corresponds to the interfacial energy between phase X and phase Y.
0=γSG – γSL – γLSCos(θC)
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 5. 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 6. 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.23 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.
Preparation of Cell-TakTM films
Cell-TakTM is a mixture of mussel foot proteins 1 and 2 in an approximate 75% to 25% ratio, respectively. The protocol used to deposit Cell-TakTMrelies upon the property of L-DOPA to come out of solution when the pH is increased. Cell-TakTM is constituted in 5% acetic acid. To prepare a film, 10 µL of a 2.36 mg/mL Cell-TakTM solution was spotted onto the substrate. To trigger binding, 10 µL of 0.2 M sodium bicarbonate was added to increase the pH to ~8-9. Films were then allowed to dry for 30 min at 37ºC and then washed with DI water. Prepared films were stored in an incubator until ready for use.
Spin coating was investigated as a new modality for depositing a protein monolayer. However, the hydrophobicity of the protein did not allow for adequate wetting of the substrates by Cell-TakTM for proper spin coating. In addition, a method used for hydrogel preparation involving incubation of a coverslip in Rain-X was tested to deposit protein layers. A coverslip was coated in Cell-TakTM and then covered by the super hydrophobic Rain-X coated coverslip. The coverslips were then pulled apart and the protein coated surface was allowed to dry. However, the consistency in thickness desired for our application made the precipitation preparation the method we chose as our standard.
Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.
Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.
