Team:Yale/Project
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<ol type="1"><li><strong>Genomically Recoded <i>E. coli</i></strong><p> | <ol type="1"><li><strong>Genomically Recoded <i>E. coli</i></strong><p> | ||
- | Whole-genome recoding was reported for the first time in 2011 by a team including our advisor, Professor Farren Isaacs, when it described replacing all TAG codons in E. coli by TAA. <sup>9</sup> Both TAG and TAA are stop codons (amber and ochre), but their transcripts are bound by different release factors (RF1 binds UAG and UAA, whereas RF2 binds UAA and UGA), not only making this recoding nonlethal but also allowing the deletion of RF1 (ΔprfA) from the recoded strain, given RF2’s response to both remaining stop codons. <p> | + | Whole-genome recoding was reported for the first time in 2011 by a team including our advisor, Professor Farren Isaacs, when it described replacing all TAG codons in <i>E. coli</i> by TAA. <sup>9</sup> Both TAG and TAA are stop codons (amber and ochre), but their transcripts are bound by different release factors (RF1 binds UAG and UAA, whereas RF2 binds UAA and UGA), not only making this recoding nonlethal but also allowing the deletion of RF1 (ΔprfA) from the recoded strain, given RF2’s response to both remaining stop codons. <p> |
This genome-wide recoding was enabled by lambda-Red recombineering<sup>10</sup> and multiplex automated genome engineering. <sup>11</sup> λ-Red recombineering uses the lambda prophage’s protein Beta to mediate the homologous recombination of an exogenous single-stranded oligonucleotide, delivered into the cell by electroporation, with the host genome, by annealing that oligonucleotide to single-stranded genomic DNA exposed on the lagging strand of the replication fork. <sup>12</sup> A 30-bp region of homology at either end of a sequence is sufficient to drive recombination of the entire strand, including mismatched regions, and mismatches thus incorporated are passed to progeny at high efficiency (up to 30%) in strains lacking methyl-directed mismatch repair (ΔmutS).<sup>13</sup> <p> | This genome-wide recoding was enabled by lambda-Red recombineering<sup>10</sup> and multiplex automated genome engineering. <sup>11</sup> λ-Red recombineering uses the lambda prophage’s protein Beta to mediate the homologous recombination of an exogenous single-stranded oligonucleotide, delivered into the cell by electroporation, with the host genome, by annealing that oligonucleotide to single-stranded genomic DNA exposed on the lagging strand of the replication fork. <sup>12</sup> A 30-bp region of homology at either end of a sequence is sufficient to drive recombination of the entire strand, including mismatched regions, and mismatches thus incorporated are passed to progeny at high efficiency (up to 30%) in strains lacking methyl-directed mismatch repair (ΔmutS).<sup>13</sup> <p> | ||
- | Multiplex automated genome engineering (MAGE) using lambda-Red recombineering can then make many directed mutations across the host genome, as was necessary to replace all 314 TAG codons in the strain EcNR2 (E. coli MG1655 ΔmutS::cat Δ(ybhB-bioAB)::[λcI857 Δ(cro-ea59)::tetR-bla]).<sup>9</sup> To allow recombineering, the lambda prophage was introduced into the host genome by P1 transduction. Because the probability of each target sequence being mutated in a given cycle is at most 30%, attempting to introduce all 314 mutations using a single pool of 314 mutagenic oligonucleotides would much sooner generate any of ~2314 other partially recoded strains before the fully recoded strain, and running enough MAGE cycles to obtain that strain would allow time for spontaneous point mutations to accumulate unchecked by the knocked-out mismatch repair system. To construct the strain we will use, then, 32 separate strains were generated, each with a different recoded sector, and those sectors were assembled by hierarchical conjugation. The recoded strain was validated by sequencing, and its RF1 was deleted to free the TAG codon for reassignment to an orthogonal translation system.<sup>14</sup> <p><lu> | + | Multiplex automated genome engineering (MAGE) using lambda-Red recombineering can then make many directed mutations across the host genome, as was necessary to replace all 314 TAG codons in the strain EcNR2 (<i>E. coli</i> MG1655 ΔmutS::cat Δ(ybhB-bioAB)::[λcI857 Δ(cro-ea59)::tetR-bla]).<sup>9</sup> To allow recombineering, the lambda prophage was introduced into the host genome by P1 transduction. Because the probability of each target sequence being mutated in a given cycle is at most 30%, attempting to introduce all 314 mutations using a single pool of 314 mutagenic oligonucleotides would much sooner generate any of ~2314 other partially recoded strains before the fully recoded strain, and running enough MAGE cycles to obtain that strain would allow time for spontaneous point mutations to accumulate unchecked by the knocked-out mismatch repair system. To construct the strain we will use, then, 32 separate strains were generated, each with a different recoded sector, and those sectors were assembled by hierarchical conjugation. The recoded strain was validated by sequencing, and its RF1 was deleted to free the TAG codon for reassignment to an orthogonal translation system.<sup>14</sup> <p><lu> |
<li><strong>Orthogonal Translation Systems</strong><p> | <li><strong>Orthogonal Translation Systems</strong><p> | ||
An amino acid can be assigned to a particular codon by a two-part translation system: a tRNA with its anticodon loop complementary to the codon, and an aminoacyl synthetase (hereafter called a synthetase) able to charge this tRNA with the amino acid. This system is orthogonal to endogenous translation systems if and only if this synthetase aminoacylates only this tRNA and endogenous synthetases cannot aminoacylate this tRNA. Usually, an unnatural amino acid thus assigned should also be sufficiently dissimilar from naturally encoded amino acids that endogenous synthetases would not charge endogenous tRNAs with the unnatural amino acid, to avoid partially reassigning other codons and disrupting cell function.<sup>15</sup> <p> | An amino acid can be assigned to a particular codon by a two-part translation system: a tRNA with its anticodon loop complementary to the codon, and an aminoacyl synthetase (hereafter called a synthetase) able to charge this tRNA with the amino acid. This system is orthogonal to endogenous translation systems if and only if this synthetase aminoacylates only this tRNA and endogenous synthetases cannot aminoacylate this tRNA. Usually, an unnatural amino acid thus assigned should also be sufficiently dissimilar from naturally encoded amino acids that endogenous synthetases would not charge endogenous tRNAs with the unnatural amino acid, to avoid partially reassigning other codons and disrupting cell function.<sup>15</sup> <p> | ||
- | Many synthetases include a nonconserved loop specific to their associated tRNA’s anticodon, preventing them from binding a tRNA reassigned to CUA, but the archaeum Methanococcus jannaschii’s tyrosyl-tRNA synthetase and its associated tRNA translation system still bind after the anticodon is mutated, and are an amber suppressor when transformed into E. coli.<sup>16</sup> This MjtRNAs was still aminoacylated by endogenous synthetases, so its specificity for its synthetase was improved by random mutation at eleven positions not directly interacting with its synthetase, followed by negative selection against nonspecific acylation and positive selection for specific acylation. <sup>17</sup> | + | Many synthetases include a nonconserved loop specific to their associated tRNA’s anticodon, preventing them from binding a tRNA reassigned to CUA, but the archaeum Methanococcus jannaschii’s tyrosyl-tRNA synthetase and its associated tRNA translation system still bind after the anticodon is mutated, and are an amber suppressor when transformed into <i>E. coli</i>.<sup>16</sup> This MjtRNAs was still aminoacylated by endogenous synthetases, so its specificity for its synthetase was improved by random mutation at eleven positions not directly interacting with its synthetase, followed by negative selection against nonspecific acylation and positive selection for specific acylation. <sup>17</sup> |
<p>This orthogonal system has since been engineered by directed evolution of its synthetase to incorporate nonstandard amino acids, in place of tyrosine. <sup>18</sup> Diverse synthetases were generated by site-directed mutagenesis of five residues in the amino acid-binding pocket, and screened by positive selection, for incorporation of the amino acid into a protein conferring antibiotic resistance, and a negative screen, against mutants resistant to the antibiotic even in the absence of the nonstandard amino acid (by treating colonies on a replica plate lacking the amino acid and then observing which were killed). Selected mutants were then recombined and subjected to further mutagenesis. <p><lu> | <p>This orthogonal system has since been engineered by directed evolution of its synthetase to incorporate nonstandard amino acids, in place of tyrosine. <sup>18</sup> Diverse synthetases were generated by site-directed mutagenesis of five residues in the amino acid-binding pocket, and screened by positive selection, for incorporation of the amino acid into a protein conferring antibiotic resistance, and a negative screen, against mutants resistant to the antibiotic even in the absence of the nonstandard amino acid (by treating colonies on a replica plate lacking the amino acid and then observing which were killed). Selected mutants were then recombined and subjected to further mutagenesis. <p><lu> | ||
<li><strong>An Improved T7 Expression System</strong> | <li><strong>An Improved T7 Expression System</strong> | ||
<p> Our first challenge in making Ampersand was to develop a system that limits basal expression of our peptide, due to its toxicity to the host. Several expression systems have been designed in the past, including the T7 promoter and T7 RNA Polymerase system, to suppress protein expression. The current BL21 (DE3) strain is leaky due to the weak suppressing promoter <i>lacUV5</i> that drives T7 RNA polymerase in the DE3 strains. As a result, low levels of toxic protein are constitutively expressed, ultimately killing the host it was made in and in turn lowering the overall yield of the protein produced.</p> | <p> Our first challenge in making Ampersand was to develop a system that limits basal expression of our peptide, due to its toxicity to the host. Several expression systems have been designed in the past, including the T7 promoter and T7 RNA Polymerase system, to suppress protein expression. The current BL21 (DE3) strain is leaky due to the weak suppressing promoter <i>lacUV5</i> that drives T7 RNA polymerase in the DE3 strains. As a result, low levels of toxic protein are constitutively expressed, ultimately killing the host it was made in and in turn lowering the overall yield of the protein produced.</p> | ||
- | <p> In order to reduce the expression of T7 RNA polymerase and create an efficient system for the expression of recombinant proteins in E. coli we chose to introduce two levels of regulation:<ul style="list-style-type:square"> <li> | + | <p> In order to reduce the expression of T7 RNA polymerase and create an efficient system for the expression of recombinant proteins in <i>E. coli</i> we chose to introduce two levels of regulation:<ul style="list-style-type:square"> <li> |
<strong>Transcriptional Regulation:</strong> We will use pZE21 of the pZ system of vectors developed by Lutz and Buschard with the P<sub>LlacO</sub> promoter to inhibit the expression of T7 RNA polymerase. | <strong>Transcriptional Regulation:</strong> We will use pZE21 of the pZ system of vectors developed by Lutz and Buschard with the P<sub>LlacO</sub> promoter to inhibit the expression of T7 RNA polymerase. | ||
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Revision as of 02:46, 18 October 2014
AMPersand: An Anti-Microbial Peptide Coating |
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The ProblemA biofilm is a community of bacteria attached to a surface that exhibits high resistance to antibiotics and human immunity. Biofilm formation poses a serious threat to the medical and shipping industries in the following ways:
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Our SolutionTo address this issue, we aimed to develop an anti-microbial adhesive peptide composed of two components. We envision these domains can be modulated to suit a variety of functional adhesive applications:
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Project Goals
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Additional Background Information
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For a detailed description of our experimental design regarding the T7 expression system, anti-microbial peptide construct, and adhesion assays, see: Materials and Methods | |||||||||||
References
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