Difference between revisions of "Team:Aachen/Lab/Methanol/Polycistronic Expression Plasmid"
Tschwanemann (Talk | contribs) (→Results) |
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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 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 a RBS in the pSB1C3 backbone [[Team:Aachen/Parts|(BBa_1585210, BBa_K1585211, BBa_K1585212, BBa_K1585213)]]. The overlapping region for the annealing of the parts were created by BsaI digest. | + | 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 a RBS in the pSB1C3 backbone [[Team:Aachen/Parts|([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# |
|} | |} | ||
Revision as of 16:01, 18 September 2015