Difference between revisions of "Part:BBa K2205018"
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===Description=== | ===Description=== | ||
| − | <p>The part BBa_K2205018 was designed as | + | <p>The part BBa_K2205018 was designed as Reporter unit of the Sensynova multicellular framework for biosensors development(Figure 1). </p> |
[[File:Framework_generic.jpg|400px|]] | [[File:Framework_generic.jpg|400px|]] | ||
| − | <p>This part was made to | + | <p>This part was made to test the modularity of the system by replacing the sfGFP reporter module of the Sensynova platform design with the chromoprotein variants spisPink ([https://parts.igem.org/Part:BBa_K1033925 BBa_K1033925]). </p> |
<p>The spisPink protein is a pink chromoprotein extracted from the coral Stylophora pistillata. It was first extracted and characterized by Alieva <i>et al</i>. under the name spisCP (GenBank: ABB17971.1) and codon optimized for <i>E. coli</i> by Genscript. The protein has an absorption maximum at 560 nm giving it a pink colour visible to the naked eye. The strong colour is readily observed in both LB or on agar plates after less than 24 hours of incubation.</p> | <p>The spisPink protein is a pink chromoprotein extracted from the coral Stylophora pistillata. It was first extracted and characterized by Alieva <i>et al</i>. under the name spisCP (GenBank: ABB17971.1) and codon optimized for <i>E. coli</i> by Genscript. The protein has an absorption maximum at 560 nm giving it a pink colour visible to the naked eye. The strong colour is readily observed in both LB or on agar plates after less than 24 hours of incubation.</p> | ||
| − | <p>The part | + | <p>The part [https://parts.igem.org/Part:BBa_K1033925 BBa_K1033925] is under the control of our connector 2, part [https://parts.igem.org/Part:BBa_K220513 BBa_K2205013], meaning it is normally repressed except in the presence of connector 2 (C12 – RHL). </p> |
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| + | [[File:Framework_Pink_Variant_Pic.jpg|400px|]] | ||
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===Characterisation=== | ===Characterisation=== | ||
| − | <p>Initial testing of | + | |
| + | <p>Initial testing of this chromoprotein reporter variant was conducted by inoculating 1ml of LB containing the antibiotic Chloramphenicol with a colony from each colour proven by colony PCR and sequencing data to be correct and grown at 37° for 2 hours as well as a control of wildtype DH5α. </p> | ||
<p>Cultures were then plated onto a LB agar + Chloramphenicol plate which were then innoculated in four locations with 10μl of connector 2 (C12 - Rhl) and grown overnight resulting in the picture below. </p> | <p>Cultures were then plated onto a LB agar + Chloramphenicol plate which were then innoculated in four locations with 10μl of connector 2 (C12 - Rhl) and grown overnight resulting in the picture below. </p> | ||
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[[File:Framework_Pink_Variant.jpg|400px|]] | [[File:Framework_Pink_Variant.jpg|400px|]] | ||
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| + | <p>In order to test this chromoprotein reporter variant into the Sensynova framework, cultures of IPTG detector, processor unit and reporter module were inoculated and grown overnight in LB+chloramphenicol (12,5ng/μl). </p> | ||
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| + | <p>The cultures were then diluted at OD600: 0,1 and mixed together to obtain co-cultures with ratio 1:1:1 and 1:1:13. Some samples were supplemented with 1mM IPTG to induce the expression of quorum sensing molecules and eventually achieve the chromoprotein visualisation (Figure 3). </p> | ||
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| + | https://static.igem.org/mediawiki/2017/3/3e/Pink_pellets2.jpg | ||
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| + | <p class="legend"><center><strong>Figure 3:</strong> Pellets collected after overnight co-cultures of IPTG + processor + Pink Chromoprotein reporter in ratios 1:1:1 and 1:1:13, with and without 1mM IPTG.</center></p> | ||
===References=== | ===References=== | ||
Latest revision as of 19:08, 1 November 2017
Sensynova Framework (Connector 2B + spisPink (Complete) - rCell)
Description
The part BBa_K2205018 was designed as Reporter unit of the Sensynova multicellular framework for biosensors development(Figure 1).
This part was made to test the modularity of the system by replacing the sfGFP reporter module of the Sensynova platform design with the chromoprotein variants spisPink (BBa_K1033925).
The spisPink protein is a pink chromoprotein extracted from the coral Stylophora pistillata. It was first extracted and characterized by Alieva et al. under the name spisCP (GenBank: ABB17971.1) and codon optimized for E. coli by Genscript. The protein has an absorption maximum at 560 nm giving it a pink colour visible to the naked eye. The strong colour is readily observed in both LB or on agar plates after less than 24 hours of incubation.
The part BBa_K1033925 is under the control of our connector 2, part BBa_K2205013, meaning it is normally repressed except in the presence of connector 2 (C12 – RHL).
Characterisation
Initial testing of this chromoprotein reporter variant was conducted by inoculating 1ml of LB containing the antibiotic Chloramphenicol with a colony from each colour proven by colony PCR and sequencing data to be correct and grown at 37° for 2 hours as well as a control of wildtype DH5α.
Cultures were then plated onto a LB agar + Chloramphenicol plate which were then innoculated in four locations with 10μl of connector 2 (C12 - Rhl) and grown overnight resulting in the picture below.
In order to test this chromoprotein reporter variant into the Sensynova framework, cultures of IPTG detector, processor unit and reporter module were inoculated and grown overnight in LB+chloramphenicol (12,5ng/μl).
The cultures were then diluted at OD600: 0,1 and mixed together to obtain co-cultures with ratio 1:1:1 and 1:1:13. Some samples were supplemented with 1mM IPTG to induce the expression of quorum sensing molecules and eventually achieve the chromoprotein visualisation (Figure 3).
References
[http://www.ncbi.nlm.nih.gov/pubmed/18648549] Alieva, Naila O., et al. "Diversity and evolution of coral fluorescent proteins." PLoS One 3.7 (2008): e2680.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 301
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 776