Team:Yale/MaterialsMethods
From 2014.igem.org
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<li><strong>Gibson Assembly of plasmids from PCR products</strong> | <li><strong>Gibson Assembly of plasmids from PCR products</strong> | ||
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Fragments were assembled into plasmids using Gibson Assembly (Gibson, Young et al. 2009). Gel purified PCR products with 5’ complimentary overhangs were | Fragments were assembled into plasmids using Gibson Assembly (Gibson, Young et al. 2009). Gel purified PCR products with 5’ complimentary overhangs were | ||
combined in equimolar ratios with ten microliters of Gibson Assembly Master Mix and additional nuclease free water to obtain a reaction volume of twenty | combined in equimolar ratios with ten microliters of Gibson Assembly Master Mix and additional nuclease free water to obtain a reaction volume of twenty | ||
microliters. The reaction was carried out in a thermocycler held constant at 50°C over the course of one hour. | microliters. The reaction was carried out in a thermocycler held constant at 50°C over the course of one hour. | ||
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- | < | + | <li><strong>Transformation of Plasmids through Electroporation</strong> |
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Transformation was carried out through electroporation. First, the plasmid to be transformed was desalted for an hour using a Millipore (type VSWP) drop | Transformation was carried out through electroporation. First, the plasmid to be transformed was desalted for an hour using a Millipore (type VSWP) drop | ||
dialysis film with 0.025 μm on ultra-pure Milli-Q water. One mL of liquid culture was centrifuged and washed twice with Milli-Q water. After washing, the | dialysis film with 0.025 μm on ultra-pure Milli-Q water. One mL of liquid culture was centrifuged and washed twice with Milli-Q water. After washing, the | ||
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immediately transferred to 1 mL of LB Lennox and placed in a 37° incubator to recover for an hour. The cells were then plated on LB Lennox agar with an | immediately transferred to 1 mL of LB Lennox and placed in a 37° incubator to recover for an hour. The cells were then plated on LB Lennox agar with an | ||
antibiotic selection factor for the transformed plasmid vector. | antibiotic selection factor for the transformed plasmid vector. | ||
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- | < | + | <li><strong>Assembly of T7 RNA Polymerase in pZE21_Y12_a12C backbone</strong> |
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Revision as of 02:50, 18 October 2014
Materials and Methods |
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T7 Riboregulation System
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Anti-Microbial Peptide ConstructConstruct Synthesis and Expression: Strains, Plasmids, and Reagents 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. 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 3).
Flag Tag Sequence: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys ![]() Figure 3. A diagram illustrating the proposed purification method. |
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Methods for Assaying Coating Adhesion Properties |
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