Plasmids

From Gary Yates, Luke Mackinder lab, University of York-UK, July 2020

Recombineering plasmid designed for generating C-terminal CrVenusYFP-3xFLAG fusion proteins.

Host strain: DB3.1 (streptomycin resistant, ccdB resistant)
Selection in E. coli: kanamycin
Selection in C. reinhardtii: hygromycin

Sequence

Emrich-Mills TZ, Yates G, Barrett J, Girr P, Grouneva I, Lau CS, Walker CE, Kwok TK, Davey JW, Johnson MP, Mackinder LCM. A recombineering pipeline to clone large and complex genes in Chlamydomonas. Plant Cell. 2021 May 31;33(4):1161-1181. doi: 10.1093/plcell/koab024. PMID: 33723601; PMCID: PMC8633747.

From Gary Yates, Luke Mackinder lab, University of York-UK, July 2020

Recombineering plasmid designed for generating C-terminal mScarlet-i-3xFLAG fusion proteins.

Host strain: DB3.1 (streptomycin resistant, ccdB resistant)
Selection in E. coli: kanamycin
Selection in C. reinhardtii: hygromycin

Sequence

Emrich-Mills TZ, Yates G, Barrett J, Girr P, Grouneva I, Lau CS, Walker CE, Kwok TK, Davey JW, Johnson MP, Mackinder LCM. A recombineering pipeline to clone large and complex genes in Chlamydomonas. Plant Cell. 2021 May 31;33(4):1161-1181. doi: 10.1093/plcell/koab024. PMID: 33723601; PMCID: PMC8633747.

From Gary Yates, Luke Mackinder lab, University of York-UK, July 2020

Recombineering plasmid designed for generating C-terminal mTurquoise2-3xHA fusion proteins.

Host strain: DB3.1 (streptomycin resistant, ccdB resistant)
Selection in E. coli: kanamycin
Selection in C. reinhardtii: zeocin

Sequence

Emrich-Mills TZ, Yates G, Barrett J, Girr P, Grouneva I, Lau CS, Walker CE, Kwok TK, Davey JW, Johnson MP, Mackinder LCM. A recombineering pipeline to clone large and complex genes in Chlamydomonas. Plant Cell. 2021 May 31;33(4):1161-1181. doi: 10.1093/plcell/koab024. PMID: 33723601; PMCID: PMC8633747.

From Anthony Berndt, Stephen Mayfield Lab, University of California, San Diego, November 2021

Vector: pUC19-based backbone
Host strain: DH5alpha
Origin: Gibson assembly using pUC19 backbone from synthesized gBlocks and elements of the pOpt vectors from the Kruse Lab.
Insert: Chloroplast directed SARS-CoV-2 Spike RBD fused to mClover fluorescent protein
Selection in E. coli: Ampicillin
Comment: Transformed into algae strain RMC1. pRMC1 map in GenBank format is also available in publication’s supplemental data.

Sequence

Berndt AJ, Smalley TN, Ren B, Simkovsky R, Badary A, Sproles AE, Fields FJ, Torres-Tiji Y, Heredia V, Mayfield SP. Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii. PLoS One. 2021 Nov 18;16(11):e0257089. doi: 10.1371/journal.pone.0257089. PMID: 34793485; PMCID: PMC8601568.

From Anthony Berndt, Stephen Mayfield Lab, University of California, San Diego, November 2021

Source: Anthony Berndt, Stephen Mayfield Lab, University of California, San Diego
Plasmid: pRMC2
Vector: pUC19-based backbone
Host strain: DH5alpha
Origin: Gibson assembly using pUC19 backbone from synthesized gBlocks and elements of the pOpt vectors from the Kruse Lab.
Insert: Secretion directed SARS-CoV-2 Spike RBD fused to mClover fluorescent protein.
Selection in E. coli: Ampicillin
Comment: Transformed in to algae strain RMC2. pRMC2 map in GenBank format is also available in publication’s supplemental data.

Sequence

Berndt AJ, Smalley TN, Ren B, Simkovsky R, Badary A, Sproles AE, Fields FJ, Torres-Tiji Y, Heredia V, Mayfield SP. Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii. PLoS One. 2021 Nov 18;16(11):e0257089. doi: 10.1371/journal.pone.0257089. PMID: 34793485; PMCID: PMC8601568.

From Anthony Berndt, Stephen Mayfield Lab, University of California, San Diego, November 2021

Source: Anthony Berndt, Stephen Mayfield Lab, University of California, San Diego
Plasmid: pRMC3
Vector: pUC19-based backbone
Host strain: DH5alpha
Origin: Gibson assembly using pUC19 backbone from synthesized gBlocks and elements of the pOpt vectors from the Kruse Lab.
Insert: ER-Golgi retained SARS-CoV-2 Spike RBD fused to mClover fluorescent protein.
Selection in E. coli: Ampicillin
Comment: Transformed in to algae strain RMC3. pRMC3 map in GenBank format also available in publication’s supplemental data.

Sequence

Berndt AJ, Smalley TN, Ren B, Simkovsky R, Badary A, Sproles AE, Fields FJ, Torres-Tiji Y, Heredia V, Mayfield SP. Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii. PLoS One. 2021 Nov 18;16(11):e0257089. doi: 10.1371/journal.pone.0257089. PMID: 34793485; PMCID: PMC8601568.