Part:BBa_K1465223
Functional carboxysome of Halothiobacillus neapolitanus with a GFP fusion under T7 control
Empty Carboxysome with GFP BBa_E0040
Usage and Biology
Naturally occuring, there are plasmids which encode different proteins of the carboxysome in bacteria. One such plasmid is <a href="2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#pHnCBS1D" target="_blank">pHnCBS1D</a> which was found in <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#H.neapolitanus" target="_blank">Halothiobacillus neapolitanus</a> (<a href="#cannon1983">Cannon and Shively, 1983</a>). We used different parts of this plasmid for the production of BioBricks. <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#H.neapolitanus" target="_blank">These BioBricks</a> (BBa_K1465202-BBa_K1465223) were used for the construction of a synthetic carboxysome-encoding plasmid. It consists up of the shell proteins CsoS1ABC and CsoS4AB and the shell associated protein CsoS2. Thereby, the proteins should form an anaerobic microcompartment. This bottom up approach allows verifying the essentiality of the different components. We used a translational fusion of one of the shell proteins CsoS1A and GFP (<a href="https://parts.igem.org/Part:BBa_K1465222" target="_blank">BBa_K1465222</a>) as an indicator of correct protein folding. A concentrated subcellular localisation of the gfp-fluorescence shows the positions of carboxysomes within the cells. This reporter function of GFP was identified and used for this purpose before (<a href="#waldo1999">Waldo et al., 1999</a>; <a href="#hsu2009">Hsu et al., 2009</a>).
The expression of <a href="https://parts.igem.org/Part:BBa_K1465223" target="_blank">BBa_K1465223</a> (pSB1C3-T7:sap-csoS4AB-csoS1CA:gfp-csoS1B) in E. coli leads to the assembly of <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Project/CO2-fixation/Carboxysome" target="_blank">carboxysomes</a>. We used a translational fusion of one shell protein (CsoS1A) with the CDS of the green fluorescent protein as an indicator of correct protein folding. The observed fluorescence in the carboxysome-expressing E. coli cells is concentrated at different points of the cell and indicates the presence of functional carboxysomes.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 216
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 374
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 882
Illegal AgeI site found at 1833
Illegal AgeI site found at 2514
Illegal AgeI site found at 3417 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 4566
Illegal SapI site found at 274
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