Plasmids used for testing and comparing the different 2A peptides were assembled using the GoldenBraid cloning system, which has been described previously [12 (link)]. Each 2A peptide used was codon optimized for expression in D. discoideum using the IDT Codon Optimization Tool (Integrated DNA Technologies), synthesized by GenScript, and then cloned into a GoldenBraid pUPD2 vector with the appropriate overhangs to act as a C-terminal linker part. Each transcriptional unit was assembled with an actin15 promoter and an actin8 terminator, and finally assembled into a backbone with a G418-resistance cassette and a D. discoideum origin of replication. We used mNeonGreen as a fluorescent protein in all constructs for comparison, mCherry as a red fluorescent protein for all western blotting experiments due to the availability of an antibody with high affinity and specificity, and mScarlet-I as a red fluorescent protein for flow cytometry because it is brighter than mCherry and thus easier to assay [33 (link)].
Plasmids for comparing dual cassette and P2A-linked antibiotic resistance gene expression were based on pDM1203 [15 (link)]. Note that as a result, all the plasmids for this set of experiments use different actin15 promoter and actin8 terminator sequences derived from this parent plasmid. To make the dual cassette plasmid pDMDC a15-mNG coaA-HygroR, a codon-optimized mNeonGreen was first cloned into the BglII/SpeI cloning site of pDM1203 and the G418-resistance cassette was swapped for a hygromycin-resistance gene by restriction digest with NheI/NotI and Gibson assembly. The hygromygin resistance fragment was obtained using pDM1501 [15 (link)] as a PCR template. In contrast to all other plasmids in this study, the resulting dual cassette plasmid is bidirectional, with the antibiotic resistance gene encoded in one direction on the plasmid and the mNeonGreen encoded in the other direction. To make the P2A-linked plasmid pDMP2A a15-mNG-P2A-HygroR, the resistance cassette was removed from pDM1203 by XhoI/BamHI digest followed by blunt-ending and religating the resultant plasmid. A codon-optimized mNeonGreen was then inserted into the BglII/SpeI site along with P2A-HygroR by Gibson assembly. The expression cassettes of the generated plasmids were verified by Sanger sequencing with GENEWIZ (Azenta Life Sciences).
Plasmids for comparing dual cassette and P2A-linked antibiotic resistance gene expression were based on pDM1203 [15 (link)]. Note that as a result, all the plasmids for this set of experiments use different actin15 promoter and actin8 terminator sequences derived from this parent plasmid. To make the dual cassette plasmid pDMDC a15-mNG coaA-HygroR, a codon-optimized mNeonGreen was first cloned into the BglII/SpeI cloning site of pDM1203 and the G418-resistance cassette was swapped for a hygromycin-resistance gene by restriction digest with NheI/NotI and Gibson assembly. The hygromygin resistance fragment was obtained using pDM1501 [15 (link)] as a PCR template. In contrast to all other plasmids in this study, the resulting dual cassette plasmid is bidirectional, with the antibiotic resistance gene encoded in one direction on the plasmid and the mNeonGreen encoded in the other direction. To make the P2A-linked plasmid pDMP2A a15-mNG-P2A-HygroR, the resistance cassette was removed from pDM1203 by XhoI/BamHI digest followed by blunt-ending and religating the resultant plasmid. A codon-optimized mNeonGreen was then inserted into the BglII/SpeI site along with P2A-HygroR by Gibson assembly. The expression cassettes of the generated plasmids were verified by Sanger sequencing with GENEWIZ (Azenta Life Sciences).
Free full text: Click here
