Difference between revisions of "Team:Aachen/Lab/Methanol/Polycistronic Expression Plasmid"
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In order to create a strain that is able to metabolize methanol, we wanted to build a polycistronic plasmid with all required genes for methanol conversion in ''E. coli''. | In order to create a strain that is able to metabolize methanol, we wanted to build a polycistronic plasmid with all required genes for methanol conversion in ''E. coli''. | ||
− | To build this multi fragment construct, the assembly method had to be chosen carefully. BioBrick Assembly is ineffective and inappropriate for such complex elements. Using [[Team:Aachen/Notebook/Protocols | + | To build this multi fragment construct, the assembly method had to be chosen carefully. BioBrick Assembly is ineffective and inappropriate for such complex elements. Using [[Team:Aachen/Notebook/Protocols#CPEC|CPEC]] or related methods, you can hardly control the order of the assembly. Thus, we had to search for an alternative that is suitable for our concept. |
{{Team:Aachen/Figure|Aachen_AP19.Poly_circular_plasmid.png|title=All methanol conversion genes in a polycistronic frame|subtitle=The general design of a polycistronic methanol uptake plasmid. | {{Team:Aachen/Figure|Aachen_AP19.Poly_circular_plasmid.png|title=All methanol conversion genes in a polycistronic frame|subtitle=The general design of a polycistronic methanol uptake plasmid. | ||
(Mdh = methanol dehydrogenase, Hps = 3-hexulose-6-phosphate, Phi = 6-phospho-3-hexuloisomerase, Xpk = phosphoketolase)|size=medium}} | (Mdh = methanol dehydrogenase, Hps = 3-hexulose-6-phosphate, Phi = 6-phospho-3-hexuloisomerase, Xpk = phosphoketolase)|size=medium}} | ||
− | To face this challenge, we used the [[Team:Aachen/Notebook/Protocols|RDP cloning standard]]. First, RDP parts out of ''mdh'', ''hps'', ''phi'' and ''xpk'' with the necessary extensions were created. | + | To face this challenge, we used the [[Team:Aachen/Notebook/Protocols#RDP_Assembly|RDP cloning standard]]. First, RDP parts out of ''mdh'', ''hps'', ''phi'' and ''xpk'' with the necessary extensions were created. |
− | These fragments were joined together with BBa_J23119 in a backbone with kanamycin resistance following the RDP Assembly method. This strong constitutive promotor from the Anderson Promoter Library was chosen because methanol conversion should be possible at every stage of bacterial growth. | + | These fragments were joined together with [http://parts.igem.org/Part:BBa_J23119 BBa_J23119] in a backbone with kanamycin resistance following the RDP Assembly method. This strong constitutive promotor from the Anderson Promoter Library was chosen because methanol conversion should be possible at every stage of bacterial growth. |
After sequence validation of the construct, the polycistronic plasmid was characterized. | After sequence validation of the construct, the polycistronic plasmid was characterized. | ||
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− | To make the coding sequences compatible with the RDP standard, they were amplified via high fidelity PCR with the primers that we designed previously. The templates were the coding sequences upstream | + | To make the coding sequences compatible with the RDP standard, they were amplified via high fidelity PCR with the primers that we designed previously. The templates were the coding sequences upstream to [http://parts.igem.org/Part:BBa_B0034 B0034] in the [http://parts.igem.org/Part:pSB1C3 pSB1C3] backbone ([http://parts.igem.org/Part:BBa_K1585210 BBa_1585210], [http://parts.igem.org/Part:BBa_K1585211 BBa_K1585211], [http://parts.igem.org/Part:BBa_K1585212 BBa_K1585212], [http://parts.igem.org/Part:BBa_K1585213 BBa_K1585213]). The overlapping region for the annealing of the parts were created by BsaI digest. |
For shorter parts like promoters, we ordered oligos that could be annealed to form short RDP parts. The fragments were mixed in an adequate ratio, and cooled down from 80 °C to room temperature in a linear gradient. | For shorter parts like promoters, we ordered oligos that could be annealed to form short RDP parts. The fragments were mixed in an adequate ratio, and cooled down from 80 °C to room temperature in a linear gradient. | ||
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! BioBrick !! corresponding RDP part !! tubefront ID | ! BioBrick !! corresponding RDP part !! tubefront ID | ||
|- | |- | ||
− | | BBa_J23119 || Z-AP19-X' || #XYD9# | + | | [http://parts.igem.org/Part:BBa_KJ23119 BBa_J23119] || Z-AP19-X' || #XYD9# |
|- | |- | ||
− | | BBa_K1585210 || X-B0034.mdh-Z'|| #PRDW# | + | | [http://parts.igem.org/Part:BBa_K1585210 BBa_K1585210] || X-B0034.mdh-Z'|| #PRDW# |
|- | |- | ||
− | | BBa_K1585211 || Z-B0034.hps-X'|| #VO4C# | + | | [http://parts.igem.org/Part:BBa_K1585211 BBa_K1585211] || Z-B0034.hps-X'|| #VO4C# |
|- | |- | ||
− | | BBa_K1585212 || X-B0034.phi-Z'|| #ZALV# | + | | [http://parts.igem.org/Part:BBa_K1585212 BBa_K1585212] || X-B0034.phi-Z'|| #ZALV# |
|- | |- | ||
− | | BBa_K1585213 + BBa_B0015 || Z-B0034.xpk.B0015-X'|| #ZR1Q# | + | | [http://parts.igem.org/Part:BBa_K1585213 BBa_K1585213] + [http://parts.igem.org/Part:BBa_B0015 BBa_B0015] || Z-B0034.xpk.B0015-X'|| #ZR1Q# |
|} | |} | ||
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− | + | {{Team:Aachen/DoubleFigure|Aachen_AP19.Poly_für_wiki.png|Aachen Poly with seq chromatogram.JPG|title1=Expression test of polycistronic plasmid on SDS-Page|title2=Successful sequencing of polycistronic construct|subtitle1=The cell pellet of ''E. coli'' BL21 Gold (DE3) that carries the polycistronic construct downstream of Anderson Promoter 19 was applied on an SDS-PAGE.}} | |
− | {{Team:Aachen/DoubleFigure| | + | |
Latest revision as of 20:46, 18 September 2015