Difference between revisions of "Team:Exeter/Lab Introduction"
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<p>We wanted to produce modified pili to primarily bind a variety of metal ions but also with reporters to allow easy verification of protein expression. A two plasmid system was designed where the modified FimH would be expressed from one plasmid and the remaining proteins in the fim operon expressed from the second plasmid. The two plasmids carried compatible origins of replication (pUC and p15A) and different antibiotic resistance genes (AmpR and CmR) to allow for co-transformation in <i>E. coli</i> (Fig.1) </p> | <p>We wanted to produce modified pili to primarily bind a variety of metal ions but also with reporters to allow easy verification of protein expression. A two plasmid system was designed where the modified FimH would be expressed from one plasmid and the remaining proteins in the fim operon expressed from the second plasmid. The two plasmids carried compatible origins of replication (pUC and p15A) and different antibiotic resistance genes (AmpR and CmR) to allow for co-transformation in <i>E. coli</i> (Fig.1) </p> | ||
<img class="rounded mx-auto d-block w-75" src="https://static.igem.org/mediawiki/2017/0/07/T--Exeter--scaledplasmids.png"> | <img class="rounded mx-auto d-block w-75" src="https://static.igem.org/mediawiki/2017/0/07/T--Exeter--scaledplasmids.png"> | ||
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<figcaption><b>Fig.1</b> Shown are two examples of the plasmids we created. On the left is the plasmid designed to produce all the proteins of the <i>fim operon</i> apart from FimH. The example specifically shown has the operon under control of an arabinose inducible promoter and is in a low copy number plasmid to avoid overexpression and metabolic burden.The plasmid on the right is an example of a FimH fusion protein under control of a rhamnose inducible promoter. This is made to produce simply the modified FimH protein with an sfGFP or metal binding domain fused at the 1st amino acid position (after the signal peptide). </figcaption> | <figcaption><b>Fig.1</b> Shown are two examples of the plasmids we created. On the left is the plasmid designed to produce all the proteins of the <i>fim operon</i> apart from FimH. The example specifically shown has the operon under control of an arabinose inducible promoter and is in a low copy number plasmid to avoid overexpression and metabolic burden.The plasmid on the right is an example of a FimH fusion protein under control of a rhamnose inducible promoter. This is made to produce simply the modified FimH protein with an sfGFP or metal binding domain fused at the 1st amino acid position (after the signal peptide). </figcaption> | ||
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<p>The FimH protein was modified to carry either sfGFP (reporter), 6xHistidine tag (reporter and metal binding) or two different metallothioneins (metal binding) (Fig.2a). See <a href="https://2017.igem.org/Team:Exeter/Basic_Part">Basic Parts</a> page for information. The Fim operon consisting of six proteins with native RBS (Fig.2b). Four different promoters were chosen to allow investigation of expression in a range of <i>E. coli</i> chassis. See <a href="https://2017.igem.org/Team:Exeter/Composite_Part">Composite Parts</a> page for information. Together this design gives a <a href="https://2017.igem.org/Team:Exeter/Part_Collection">collection of parts</a> for modified pili production. </p> | <p>The FimH protein was modified to carry either sfGFP (reporter), 6xHistidine tag (reporter and metal binding) or two different metallothioneins (metal binding) (Fig.2a). See <a href="https://2017.igem.org/Team:Exeter/Basic_Part">Basic Parts</a> page for information. The Fim operon consisting of six proteins with native RBS (Fig.2b). Four different promoters were chosen to allow investigation of expression in a range of <i>E. coli</i> chassis. See <a href="https://2017.igem.org/Team:Exeter/Composite_Part">Composite Parts</a> page for information. Together this design gives a <a href="https://2017.igem.org/Team:Exeter/Part_Collection">collection of parts</a> for modified pili production. </p> | ||
Revision as of 09:45, 1 November 2017
Pili+
Modified pili expression
We wanted to produce modified pili to primarily bind a variety of metal ions but also with reporters to allow easy verification of protein expression. A two plasmid system was designed where the modified FimH would be expressed from one plasmid and the remaining proteins in the fim operon expressed from the second plasmid. The two plasmids carried compatible origins of replication (pUC and p15A) and different antibiotic resistance genes (AmpR and CmR) to allow for co-transformation in E. coli (Fig.1)
The FimH protein was modified to carry either sfGFP (reporter), 6xHistidine tag (reporter and metal binding) or two different metallothioneins (metal binding) (Fig.2a). See Basic Parts page for information. The Fim operon consisting of six proteins with native RBS (Fig.2b). Four different promoters were chosen to allow investigation of expression in a range of E. coli chassis. See Composite Parts page for information. Together this design gives a collection of parts for modified pili production.
