Team:Calgary/Project/DataPage

From 2013.igem.org

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<h2>Characterization of new parts submitted to the Registry</h2>
<h2>Characterization of new parts submitted to the Registry</h2>
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<p> <ul>This year we submitted two new reporters to the registry, <b>Beta-lactamase</b> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189008">Bba_ K118908</a>) and <b>Recombinant Human Ferritin</b> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189008">Bba_ K118918</a>). We built the parts, expressed, and purified these proteins < show here>. </ul></p>
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<ul>This year we submitted two new reporters to the registry, <b>Beta-lactamase</b> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189008">Bba_ K118908</a>) and <b>Recombinant Human Ferritin</b> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189008">Bba_ K118918</a>). We built the parts, expressed, and purified these proteins < show here>. </ul>
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<ul> Firstly, we converted ferritin into Prussian blue ferritin, which has high catalytic activity and then characterized it as a reporter. We tested Prussian Blue ferritin using  two different substrates, ABTS and TMB, a range of temperatures and pH conditions. We did a Michelis-Menten kinetic analysis with Prussian blue ferritin and characterized it under ranging temperature and pH conditions.</ul>
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<p><ul> Firstly, we converted ferritin into Prussian blue ferritin, which has high catalytic activity and then characterized it as a reporter. We tested Prussian Blue ferritin using  two different substrates, ABTS and TMB, a range of temperatures and pH conditions. We did a Michelis-Menten kinetic analysis with Prussian blue ferritin and characterized it under ranging temperature and pH conditions.</ul>
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<ul> We also showed that Prussian blue ferritin is a feasible reporter in our system by doing dot-blots on nitrocellulose and our model prototype (see below) with its substrates to give us a quantitative and qualitative output, respectively. In addition, we showed that ferritin, a protein with 24 subunits is expressed and functional in our system.</ul>
<ul> We also showed that Prussian blue ferritin is a feasible reporter in our system by doing dot-blots on nitrocellulose and our model prototype (see below) with its substrates to give us a quantitative and qualitative output, respectively. In addition, we showed that ferritin, a protein with 24 subunits is expressed and functional in our system.</ul>
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<ul> We have also expressed, isolated and converted human recombinant ferritin into Prussian blue ferritin on which we have done initial characterization on nitrocellulose. <LINK TO DATA PLEASE></ul></p>
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<ul> We have also expressed, isolated and converted human recombinant ferritin into Prussian blue ferritin on which we have done initial characterization on nitrocellulose. <LINK TO DATA PLEASE></ul>
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<ul>We have cloned, expressed and purified beta lactamase in our bacterial cells which we have characterized using an ampicillin <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Reporter/BetaLactamase">survival assays</a>. </ul>
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<p> <ul>We have cloned, expressed and purified beta lactamase in our bacterial cells which we have characterized using an ampicillin <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Reporter/BetaLactamase">survival assays</a>. </ul>
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<ul>We have also built the TALE A linked to beta lactamase, representing the alternative mobile unit in our sensor, which we have expressed and purified. We characterized this fusion protein with an ampicillin assay <link>. We have also done initial characterizations of the beta lactamase as a reporter by using benzylpenicillin as a substrate which gives a pH output. When combined with phenol red as a pH indicator, we have shown a colourimetric output which correlates to changing amounts of beta lactamase. </ul> <link>.  
<ul>We have also built the TALE A linked to beta lactamase, representing the alternative mobile unit in our sensor, which we have expressed and purified. We characterized this fusion protein with an ampicillin assay <link>. We have also done initial characterizations of the beta lactamase as a reporter by using benzylpenicillin as a substrate which gives a pH output. When combined with phenol red as a pH indicator, we have shown a colourimetric output which correlates to changing amounts of beta lactamase. </ul> <link>.  

Revision as of 21:19, 27 September 2013

Data Page

Characterization of new parts submitted to the Registry

    This year we submitted two new reporters to the registry, Beta-lactamase (Bba_ K118908) and Recombinant Human Ferritin (Bba_ K118918). We built the parts, expressed, and purified these proteins < show here>.
    Firstly, we converted ferritin into Prussian blue ferritin, which has high catalytic activity and then characterized it as a reporter. We tested Prussian Blue ferritin using two different substrates, ABTS and TMB, a range of temperatures and pH conditions. We did a Michelis-Menten kinetic analysis with Prussian blue ferritin and characterized it under ranging temperature and pH conditions.
    We also showed that Prussian blue ferritin is a feasible reporter in our system by doing dot-blots on nitrocellulose and our model prototype (see below) with its substrates to give us a quantitative and qualitative output, respectively. In addition, we showed that ferritin, a protein with 24 subunits is expressed and functional in our system.
    We have also expressed, isolated and converted human recombinant ferritin into Prussian blue ferritin on which we have done initial characterization on nitrocellulose.
    We have cloned, expressed and purified beta lactamase in our bacterial cells which we have characterized using an ampicillin survival assays.
    We have also built the TALE A linked to beta lactamase, representing the alternative mobile unit in our sensor, which we have expressed and purified. We characterized this fusion protein with an ampicillin assay . We have also done initial characterizations of the beta lactamase as a reporter by using benzylpenicillin as a substrate which gives a pH output. When combined with phenol red as a pH indicator, we have shown a colourimetric output which correlates to changing amounts of beta lactamase.
.

Further characterization and improvement of parts already present within the registry

  • The kozak sequence on TALE A ( Part:BBa_K782004) and TALE B ( Part:BBa_K782006) were removed, and an IPTG inducible promoter with RBS ( Part:BBa_J04500) was constructed upstream of the genes, so that the part could be expressed in E.coli
    • . We submitted these parts as J04500+His+TALA ( BBa_K1189000) and J04500+His +TALB ( BBa_K1189001).

    We made further improvement to TALE A ( Part:BBa_K782004) and TALE B ( Part:BBa_K782006) in our bigger constructs including J04500+His+TALEA+link+Kcoil ( BBa_K1189029), J04500+His+TALB+link+Kcoil ( BBa_K1189030), J04500+His+TALA+Link+Blac ( BBa_K1189031), and J04500+His+TALA+hFTN+lFTN ( BBa_K1189021):

  • They were codon optimized for E.coli to increase the expression level in a chassis used by most of the iGEM community.
  • the Nuclear Localization Sequence (NLS) at the C-teminus of the TALEs were removed, as E.coli does not have a nucleus.
  • a KasI restriction cut site was added after the C-terminus of the TALE, in order to create a plug-and-play system. If a future iGEM team requires to use one of our parts in the future, they can easily swap TALE A and TALE B with their own engineered TALE.
  • Slovenia 2012 iGEM team conducted all their assays in vivo. We were able to show that TALE A and TALE B can be purified. The following constructs were successfully expressed and purified: J04500+His+TALA ( BBa_K1189000); J04500+His +TALB ( BBa_K1189001); J04500+His+TALEA+link+Kcoil ( BBa_K1189029); J04500+His+TALA+Link+Blac ( BBa_K1189031); J04500+His+TALA+hFTN+lFTN ( BBa_K1189021):

    .

    Slovenia 2012 iGEM team did a great characterization of TALE A ( Part:BBa_K782004) and TALE B ( Part:BBa_K782006). We used these parts as a proof of concept of our system. Along the way we made some changes to these parts to make them more useful to us and the parts registry. Slovenia 2012 iGEM team used these parts in eukaryotic cells. Therefore, these parts had a kozak sequence before the start of the gene.

    In 2012, the Wagenigen team worked with and did initial characterized a coiled coil system known as the E and K coils for their project. However, they did not submit the physical DNA for the coils. These coiled coils are very useful for in vitro assembly of different proteins and their ability to scaffold proteins together when fusion does not work, making an attractive addition to the registry. We submitted these parts K coil(Bba_K1189027) and E coil (Bba_K1189011). We also added these parts with a his-6 tag such that fusion proteins with K or E coil can be purified out using affinity chromatography. These four parts have been submitted in the Freiburg fusion biobrick backbone to allow easier construction of fusion protein for future teams. We have done initial characterization demonstrating that the coils allow in vitro assembly by doing a dot blot assay with our mobile TALE detector and Prussian blue reporter, both of which we have built, expressed, and purified with the coiled coils.

    We have also mutated, optimized, and repurposed Slovenia’s TALE proteins from 2012 to work in E. coli . The TALE that Slovenia submitted had mutations, as it is difficult to sequence RVDs in TALEs. We amended the TALE to fit the correct binding sequence. This was not optimized to work in E. coli, it had a eukaryotic ribosomal binding site called a Kozak sequence. In the presence ok Kozak sequence the TALEs are not expressed in E. coli . We removed the Kozak sequence and codon optimized the TALEs for expression in E. coli. Further, we characterized the TALEs and showed that they bind to the correct nucleotide sequences.

    Something about the kas1 site that ALi put in

    Modelleing

    Quantitative and qualitative

    Collaboration

    Sensigem

    Additional Work and Characterization

    We characterized a portable prototype showing that this final system is feasible. We used a homestyle pregnancy kit to show that this is possible LINK HERE. We showed that it is possible to flow DNA through the strip, add protein on the strip and get a colour output using ferritin.

    Human practices

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