Team:Yale/Project
From 2014.igem.org
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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 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> | ||
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 | + | 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> |
<li><strong>Background: Orthogonal translation systems</strong><p> | <li><strong>Background: Orthogonal translation systems</strong><p> | ||
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<p> | <p> | ||
- | In summary, the benefits to this type of system are its robustness, portability, and efficiency in that it does not require the cell to expend more energy on the constitutive synthesis of another protein. We theorize 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 and permit stable cloning of toxic recombinant proteins. | + | In summary, the benefits to this type of system are its robustness, portability, and efficiency in that it does not require the cell to expend more energy on the constitutive synthesis of another protein. We theorize 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 and permit stable cloning of toxic recombinant proteins.<br> |
<center><img src="https://static.igem.org/mediawiki/2014/e/ed/T7_Diagram_For_Wiki.png"></center> | <center><img src="https://static.igem.org/mediawiki/2014/e/ed/T7_Diagram_For_Wiki.png"></center> | ||
- | <p> | + | <p><br> |
<center> | <center> | ||
<i><strong>Figure X.</strong> This diagram illustrates the T7 riboregulation system developed to ensure low basal levels of protein expression.<sup>19</sup> </i></center><p> | <i><strong>Figure X.</strong> This diagram illustrates the T7 riboregulation system developed to ensure low basal levels of protein expression.<sup>19</sup> </i></center><p> |
Revision as of 00:52, 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 exhibit 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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How it Works
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Materials and Methods | |||||||||||
Results
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References
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