Team:Yale/Results

From 2014.igem.org

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<h1>T7 Riboregulation System</h1>
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<ol type="I"><li><strong>T7 Riboregulation System: Experimental Design</strong><ul style="list-style-type:square">  
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<li><strong>Strains, Plasmids, and Reagents:</strong>
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<ol type="I">
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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.
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<p>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.</p>
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<li><strong>T7 RNA polymerase design and creation</strong>
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<p>
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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.
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<br>
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<center><img style='border:2px solid #000000'  src="https://static.igem.org/mediawiki/2014/6/66/Yale_figure7.png"></center>
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<p>
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<center>
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<i><strong>Figure 1.</strong> Preliminary gel screening of Mach 1 strains containing transformed pZE21_A12C_T7RNA plasmids created via Gibson Assembly. Used combinations of general pZE21 sequencing primers, F: CAGGGCTTCCCAACCTTAC, R: CGCCTTTGAGTGAGCTGATA, and internal T7 primers, F: TCCCTTACAACATGGACTGGC, R: CCCACCAAGTGTTCTCCAG. The corresponding sizes are labelled on the side. The negative control for the external primers is the ancestor plasmid, which contains chloramphenicol acetyl transferase (CAT) instead of T7, and is 1.1 kb instead of 3.3 kb.</i></center><br></p>
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<li><strong>Two Levels of Regulation for T7 Polymerase Expression:</strong> The P<sub>LlacO</sub> 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).
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<p>A second promoter, P<sub>LtetO</sub>, 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 <i>E. coli</i> 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.
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<p>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 <i>E. coli</i> 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.</p></ul>
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<li><strong>Adhesion Testing</strong>: <ul><li><strong>Mass Retention of MAPs Under Stress</strong>
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<p>
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<p>Preliminary proof of concept testing was conducted on a commercially available MAP-based product known as Cell-Tak <sup>TM</sup>. Cell-Tak<sup>TM</sup> 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 H<sub>2</sub>O 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 H<sub>2</sub>O 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.
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<center>
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<i><strong>Figure 2.</strong> Sequencing data for T7 RNA polymerase construct, using the general pZE21 sequencing primers, which amplify upstream of the taRNA and crRNA system. Sequencing done via Keck Biotechnology Resource Laboratory. Gray indicates consensus with the desired sequence. Image made using geneious.</i></center><br></p>
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<p>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.</p>
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<li><strong>Functional Assays for Riboregulated T7 system</strong>  
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<p>
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Experimental plan for the GFP fluorescence assay testing the efficacy of the T7 riboregulation system. The T7 riboregulation system, pZE21_A12C_T7RNA, would express sfGFP behind a T7 promoter, in the plasmid pZA21. Either plasmid, and both plasmids together, were transformed into ECNR2 and induced with either IPTG and ATC. ECNR2 is the ancestral strain. A positive control was the same pZA21_T7sfGFP plasmid in ECNR2, and the same T7 RNA polymerase gene inserted in a regular pZE21 plasmid with a pLtetO promoter, and a negative control with the pZA21_T7sfGFP in ECNR2 without any plasmid that contains T7 RNA. </p> <br>
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<center><img style='border:2px solid #000000' src="https://static.igem.org/mediawiki/2014/f/ff/Yale_figure8.png"></center>
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<i><strong>Figure 3.</strong> The functionalities behind the GFP assay as described above.</i></center><br></p>
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<center><img style='border:2px solid #000000' src="https://static.igem.org/mediawiki/2014/0/03/Yale_figure9.png"></center>
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<p>
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<center>
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<i><strong>Figure 4.</strong> Conformational assay to test the functionality of the pZA21_T7sfGFP, which is sfGFP placed behind the T7 promoter. The plasmid was transformed into a BL21(DE3) strain, which constitutively expresses T7 RNA polymerase. The strain, as well as untransformed BL21(DE3), were grown overnight and assayed using a Synergy H1 Biotek Platereader. Fluorescence measurement was taken by exciting the cells at 485 nm and detecting at 528 nm, with a bandpass of 4 nm on each side. The optical density was also taken at 600 nm, and the fluorescence data was normalized by dividing fluorescence by optical density. What is shown is the average of 4 replicates each. </i></center></p><br>
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<tr class="tableizer-firstrow"><th>Plasmid</th><th>Antibiotic Marker</th><th>Inducer Conditions 1</th><th>Inducer Conditions 2</th><th>Inducer Conditions 3</th><th>Inducer Conditions 4</th></tr>
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<tr><td>pZE21_A12C_T7RNA, pZA21_T7sfGFP</td><td>Kanamycin, Spectinomycin</td><td>ATC, IPTG</td><td>ATC only</td><td>IPTG only</td><td>No Inducers</td></tr>
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<tr><td>pZE21_A12C_T7RNA</td><td>Kanamycin</td><td>ATC, IPTG</td><td>ATC only</td><td>IPTG only</td><td>No Inducers</td></tr>
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<tr><td>pZA21_T7sfGFP</td><td>Spectinomycin</td><td>ATC, IPTG</td><td>ATC only</td><td>IPTG only</td><td>No Inducers</td></tr>
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<tr><td>ECNR2, no plasmids</td><td>None</td><td>ATC, IPTG</td><td>ATC only</td><td>IPTG only</td><td>No Inducers</td></tr>
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</table>
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<p>
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<center>
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<i><strong>Table 1.</strong> Experimental conditions for the GFP assay. Each plasmid combination was paired with each inducer combination, and the conditions were made in six replicates.</i></center></p>
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<div class = "tinycall">
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<h1>Ampersand Construct </h1>
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</div>
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<div class = "well">
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<ol type="I">
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<li><strong>Synthesis and Transformation</strong>
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<p>The AMP-MAP construct, also known as Ampersand construct, was received and cloned into a standard pZE21 plasmid backbone with pLtetO promoter, as the T7 Riboregulation system was incomplete at the time. The construct has been sent for sequencing, and is now awaiting functional assays.
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<center><img src="https://static.igem.org/mediawiki/2014/4/42/Yale_construct_sequencing.jpg" height = 300 width = auto></center>
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<p>
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                <center>
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                <i><strong>Figure 5.</strong>  Gel results of the Gibson assembly product of the construct into pZE21 plasmid backbone, with a co-transformed OTS system. Used same universal primers as before, and the results present a difficulty in sequencing: lanes 3 and 4 are the Tyrosine suppressor system WITHOUT the construct, which means sequencing data are unable to be obtained unless performed on a strain without the OTS, which unfortunately interfere with the functional assays planned.</i></center></p>
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<li><strong> Functional Assay</strong><p>
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Functional assays are ongoing and results will be presented at the jamboree.</p>
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<td colspan="12">
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<div class = "tinycall">
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<h1>Adhesion Testing </h1>
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</div>
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<div class = "well">
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<ol type="I">
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<li><strong>Mass Retention of Mussel Adhesion Proteins (MAPs) Under Stress</strong>
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<p>Preliminary proof of concept testing was conducted on a commercially available MAP-based product known as Cell-Tak <sup>TM</sup>. Cell-Tak<sup>TM</sup> 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 H<sub>2</sub>O 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 microbalance (Mettler Toledo MX5) 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 H<sub>2</sub>O 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.
<center><img src="https://static.igem.org/mediawiki/2014/a/aa/Erosion_Fig_iGEMwiki.png" height = 300 width = auto></center>
<center><img src="https://static.igem.org/mediawiki/2014/a/aa/Erosion_Fig_iGEMwiki.png" height = 300 width = auto></center>
<p>
<p>
                 <center>
                 <center>
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                 <i><strong>Figure X.</strong> <strong>(A)</strong> The erosion of Cell-Tak <sup>TM</sup> under conditions of DI water erosion. <strong>(B)</strong> The erosion of Cell-Tak <sup>TM</sup> under conditions of 5% acetic acid erosion.</i></center></p>
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                 <i><strong>Figure 5.</strong> <strong>(A)</strong> The erosion of Cell-Tak <sup>TM</sup> under conditions of DI water erosion. <strong>(B)</strong> The erosion of Cell-Tak <sup>TM</sup> under conditions of 5% acetic acid erosion.</i></center></p>
<li><strong> Determination of Surface Energies of MAP Films</strong><p>
<li><strong> Determination of Surface Energies of MAP Films</strong><p>
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A contact angle measurement of a Cell-Tak<sup>TM</sup> 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 H<sub>2</sub>O. 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 resistance to water in order to survive and anchor themselves in constantly wet environments.  
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A contact angle measurement of a Cell-Tak<sup>TM</sup> 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 H<sub>2</sub>O. 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.  
<center><img src="https://static.igem.org/mediawiki/2014/8/86/IGEM_Contact_Angle_Data_wiki.png" height = 300 width = auto></center>
<center><img src="https://static.igem.org/mediawiki/2014/8/86/IGEM_Contact_Angle_Data_wiki.png" height = 300 width = auto></center>
<p>
<p>
                 <center>
                 <center>
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                 <i><strong>Figure X.</strong><strong>(A)</strong> The profile photograph of a drop on an untreated silica substrate used for contact angle determination. <strong>(B)</strong> The profile photograph of a Cell-Tak <sup>TM</sup> treated surface used for contact angle determination.</i></center></p>
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                 <i><strong>Figure 6.</strong><strong> (A)</strong> The profile photograph of a drop on an untreated silica substrate used for contact angle determination. <strong>(B)</strong> The profile photograph of a Cell-Tak <sup>TM</sup> treated surface used for contact angle determination.</i></center></p>

Latest revision as of 03:53, 18 October 2014

Results

T7 Riboregulation System

  1. T7 RNA polymerase design and creation

    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.

    Figure 1. Preliminary gel screening of Mach 1 strains containing transformed pZE21_A12C_T7RNA plasmids created via Gibson Assembly. Used combinations of general pZE21 sequencing primers, F: CAGGGCTTCCCAACCTTAC, R: CGCCTTTGAGTGAGCTGATA, and internal T7 primers, F: TCCCTTACAACATGGACTGGC, R: CCCACCAAGTGTTCTCCAG. The corresponding sizes are labelled on the side. The negative control for the external primers is the ancestor plasmid, which contains chloramphenicol acetyl transferase (CAT) instead of T7, and is 1.1 kb instead of 3.3 kb.

    Figure 2. Sequencing data for T7 RNA polymerase construct, using the general pZE21 sequencing primers, which amplify upstream of the taRNA and crRNA system. Sequencing done via Keck Biotechnology Resource Laboratory. Gray indicates consensus with the desired sequence. Image made using geneious.

    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.

  2. Functional Assays for Riboregulated T7 system

    Experimental plan for the GFP fluorescence assay testing the efficacy of the T7 riboregulation system. The T7 riboregulation system, pZE21_A12C_T7RNA, would express sfGFP behind a T7 promoter, in the plasmid pZA21. Either plasmid, and both plasmids together, were transformed into ECNR2 and induced with either IPTG and ATC. ECNR2 is the ancestral strain. A positive control was the same pZA21_T7sfGFP plasmid in ECNR2, and the same T7 RNA polymerase gene inserted in a regular pZE21 plasmid with a pLtetO promoter, and a negative control with the pZA21_T7sfGFP in ECNR2 without any plasmid that contains T7 RNA.


    Figure 3. The functionalities behind the GFP assay as described above.

    Figure 4. Conformational assay to test the functionality of the pZA21_T7sfGFP, which is sfGFP placed behind the T7 promoter. The plasmid was transformed into a BL21(DE3) strain, which constitutively expresses T7 RNA polymerase. The strain, as well as untransformed BL21(DE3), were grown overnight and assayed using a Synergy H1 Biotek Platereader. Fluorescence measurement was taken by exciting the cells at 485 nm and detecting at 528 nm, with a bandpass of 4 nm on each side. The optical density was also taken at 600 nm, and the fluorescence data was normalized by dividing fluorescence by optical density. What is shown is the average of 4 replicates each.


    PlasmidAntibiotic MarkerInducer Conditions 1Inducer Conditions 2Inducer Conditions 3Inducer Conditions 4
    pZE21_A12C_T7RNA, pZA21_T7sfGFPKanamycin, SpectinomycinATC, IPTGATC onlyIPTG onlyNo Inducers
    pZE21_A12C_T7RNAKanamycinATC, IPTGATC onlyIPTG onlyNo Inducers
    pZA21_T7sfGFPSpectinomycinATC, IPTGATC onlyIPTG onlyNo Inducers
    ECNR2, no plasmidsNoneATC, IPTGATC onlyIPTG onlyNo Inducers

    Table 1. Experimental conditions for the GFP assay. Each plasmid combination was paired with each inducer combination, and the conditions were made in six replicates.

Ampersand Construct

  1. Synthesis and Transformation

    The AMP-MAP construct, also known as Ampersand construct, was received and cloned into a standard pZE21 plasmid backbone with pLtetO promoter, as the T7 Riboregulation system was incomplete at the time. The construct has been sent for sequencing, and is now awaiting functional assays.

    Figure 5. Gel results of the Gibson assembly product of the construct into pZE21 plasmid backbone, with a co-transformed OTS system. Used same universal primers as before, and the results present a difficulty in sequencing: lanes 3 and 4 are the Tyrosine suppressor system WITHOUT the construct, which means sequencing data are unable to be obtained unless performed on a strain without the OTS, which unfortunately interfere with the functional assays planned.

  2. Functional Assay

    Functional assays are ongoing and results will be presented at the jamboree.

Adhesion Testing

  1. Mass Retention of Mussel Adhesion Proteins (MAPs) Under Stress

    Preliminary proof of concept testing was conducted on a commercially available MAP-based product known as Cell-Tak TM. Cell-TakTM 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 microbalance (Mettler Toledo MX5) 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.

    Figure 5. (A) The erosion of Cell-Tak TM under conditions of DI water erosion. (B) The erosion of Cell-Tak TM under conditions of 5% acetic acid erosion.

  2. Determination of Surface Energies of MAP Films

    A contact angle measurement of a Cell-TakTM 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.

    Figure 6. (A) The profile photograph of a drop on an untreated silica substrate used for contact angle determination. (B) The profile photograph of a Cell-Tak TM treated surface used for contact angle determination.

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