Plasmids

From Beatriz Cruz, Stephen Mayfield Lab, University California-San Diego, February 2015

host strain: DH10
amp resistant

Sequence (.gb file)

Rasala BA, Barrera DJ, Ng J, Plucinak TM, Rosenberg JN, Weeks DP, Oyler GA, Peterson TC, Haerizadeh F, Mayfield SP (2013) Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii. Plant J 74:545-56

From Beatriz Cruz, Stephen Mayfield Lab, University California-San Diego, February 2015

host strain: DH10
amp resistant

Sequence (.gb file)

Rasala BA, Barrera DJ, Ng J, Plucinak TM, Rosenberg JN, Weeks DP, Oyler GA, Peterson TC, Haerizadeh F, Mayfield SP (2013) Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii. Plant J 74:545-56

From Beatriz Cruz, Stephen Mayfield Lab, University California-San Diego, February 2015

host strain: DH10
amp resistant

Sequence (.gb file)

Rasala BA, Barrera DJ, Ng J, Plucinak TM, Rosenberg JN, Weeks DP, Oyler GA, Peterson TC, Haerizadeh F, Mayfield SP (2013) Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii. Plant J 74:545-56

From Beatriz Cruz, Stephen Mayfield Lab, University California-San Diego, February 2015

host strain: DH10
amp resistant

Sequence (.gb file)

Rasala BA, Barrera DJ, Ng J, Plucinak TM, Rosenberg JN, Weeks DP, Oyler GA, Peterson TC, Haerizadeh F, Mayfield SP (2013) Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii. Plant J 74:545-56

From Beatriz Cruz, Stephen Mayfield Lab, University California-San Diego, February 2015

Transformation vector that enables protein targeting to the nucleus.

host strain: DH10
amp resistant

Sequence (.gb file)

Rasala BA, Chao SS, Pier M, Barrera DJ, Mayfield SP (2014) Enhanced genetic tools for engineering multigene traits into green algae. PLoS One 9:e94028

From Beatriz Cruz, Stephen Mayfield Lab, University California-San Diego, February 2015

Transformation vector that enables protein targeting to the mitochondria.

host strain: DH10
amp resistant

Sequence (.gb file)

Rasala BA, Chao SS, Pier M, Barrera DJ, Mayfield SP (2014) Enhanced genetic tools for engineering multigene traits into green algae. PLoS One 9:e94028

From Beatriz Cruz, Stephen Mayfield Lab, University California-San Diego, February 2015

Transformation vector that enables protein targeting to the endoplasmic reticulum (ER).

host strain: DH10
amp resistant

Sequence (.gb file)

Rasala BA, Chao SS, Pier M, Barrera DJ, Mayfield SP (2014) Enhanced genetic tools for engineering multigene traits into green algae. PLoS One 9:e94028

From Beatriz Cruz, Stephen Mayfield Lab, University California-San Diego, February 2015

Transformation vector that enables protein targeting to the endoplasmic reticulum (ER).

host strain: DH10
amp resistant

Sequence (.gb)

Rasala BA, Chao SS, Pier M, Barrera DJ, Mayfield SP (2014) Enhanced genetic tools for engineering multigene traits into green algae. PLoS One 9:e94028

From Beatriz Cruz, Stephen Mayfield Lab, University California-San Diego, February 2015

Transformation vector that enables protein targeting to the chloroplast.

host strain: DH10
amp resistant

Sequence (.gb file)

Rasala BA, Chao SS, Pier M, Barrera DJ, Mayfield SP (2014) Enhanced genetic tools for engineering multigene traits into green algae. PLoS One 9:e94028

From Marilyn Kobayashi, Niyogi lab, University of California-Berkeley, April 2015

Sequence (SeqBuilder)

Sequence  (MS Word)

Map

Depège N, Bellafiore S, Rochaix JD. Role of chloroplast protein kinase Stt7 in LHCII phosphorylation and state transition in Chlamydomonas. Science. 2003 Mar 7;299(5612):1572-5. doi: 10.1126/science.1081397. PMID: 12624266.

From Sergio García and Guy Cardineau, Tecnológico de Monterrey, Campus Monterrey-Mexico, May 2015

The construct was generated by Polymerase Chain Reaction, amplifying the open reading frame of tetX from the BtetX plasmid with a proof-reading high fidelity (1 error/100,000 bp = 0.001%) enzyme and oligos that carried XhoI and BamHI sites in their 5’ends. The amplicon was digested with BamHI and XhoI, and cloned into the corresponding sites of plasmid pHsp70A/RbcS2-cgLuc, replacing the luciferase ORF with that of tetX.

Comment: The AtetX plasmid can be used to select nuclear transformants of Chlamydomonas reinhardtii. It has been proven to work in the cell wall deficient strain CC-849, however the wild-type cell-walled strain CC-125 is as sensitive to tetracycline as strain CC-849. The transformants should be selected in in TAP plates containing 15 µg/mL of tetracycline and maintained below a light intensity of 24 µmoles m-2 s-1. Transformed colonies will be visible after 8 days and can resist up to 100 µg/mL of tetracycline.

host strain: DH5α
amp resistant

Map and sequence

From Sergio García and Guy Cardineau, Tecnológico de Monterrey, Campus Monterrey-Mexico, May 2015

The TetX open reading frame [Genbank: JQ990987] was synthesized de novo with codons optimized for Chlamydomonas reinhardtii cytoplasmic expression under the control of constitutive beta 2 tubulin promoter and chlamyopsin1 3’UTR.

Comment: The BtetX plasmid can be used to select nuclear transformants of Chlamydomonas reinhardtii. It has been proven to work in the cell wall deficient strain CC-849, however the wild-type cell-walled strain CC-125 is as sensitive to tetracycline as strain CC-849. The transformants should be selected in TAP plates containing 15 µg/mL of tetracycline and maintained below a light intensity of 24 µmoles m-2 s-1. Transformed colonies will be visible after 8 days and can resist up to 100 µg/mL of tetracycline.

host strain: DH5α
amp resistant

Map and sequence

From Rosanna Young, Saul Purton lab, University College London, UK, October 2015

CrCD is a conditional negative selectable marker for use in the C. reinhardtii chloroplast that confers sensitivity to 5-fluorocytosine by converting it to toxic 5-fluorouracil (Young and Purton 2014). Plasmid pRY127d contains the crCD gene, with a C-terminal HA tag, inserted into the chloroplast expression vector pSRSapI. CrCD is a modified and codon-optimised version of the E. coli cytosine deaminase (codA) gene. The crCD gene in pRY127d uses the C. reinhardtii psaA exon 1 promoter and 5′ UTR and the C. reinhardtii rbcL 3′ UTR; the flanking regions in the construct target the crCD cassette downstream of psbH by homologous recombination.

This plasmid is called pCD in Young and Purton 2015.

Sequence (.docx file)

From Don Weeks, University of Nebraska via David Wright, Spalding lab, Iowa State University, February 2016

host strain: DH10B
amp resistant

Sequence

Plucinak TM, Horken KM, Jiang W, Fostvedt J, Nguyen ST, Weeks DP (2015) Improved and versatile viral 2A platforms for dependable and inducible high-level expression of dicistronic nuclear genes in Chlamydomonas reinhardtii. Plant J. 82:717-29

From Don Weeks, University of Nebraska via David Wright, Spalding lab, Iowa State University, February 2016

host strain: DH10B
amp resistant

Sequence

Plucinak TM, Horken KM, Jiang W, Fostvedt J, Nguyen ST, Weeks DP (2015) Improved and versatile viral 2A platforms for dependable and inducible high-level expression of dicistronic nuclear genes in Chlamydomonas reinhardtii. Plant J. 82:717-29

From Don Weeks, University of Nebraska via David Wright, Spalding lab, Iowa State University, February 2016

host strain: DH10B
amp resistant

Sequence

Plucinak TM, Horken KM, Jiang W, Fostvedt J, Nguyen ST, Weeks DP (2015) Improved and versatile viral 2A platforms for dependable and inducible high-level expression of dicistronic nuclear genes in Chlamydomonas reinhardtii. Plant J. 82:717-29

From Hussam Hassan Nour-Eldin, Stephen Mayfield Lab, UC San Diego, November 2015

Insert: ARG7 promoter/5’ UTR:ARG7 coding sequence:ARG7 3’ UTR. Total insert size is 3,479 bp.

This plasmid can transform the Chlamydomonas nuclear genome in strains that are deficient or disrupted at the ARG7 locus, and therefore are arginine auxotrophic. This plasmid exhibits much greater transformation efficiency than plasmids with the genomic ARG7 fragment.

USER (uracil-specific excision reagent) fusion cloning was used to construct this vector. The ARG7 promoter and 5’ UTR, the ARG7 coding sequence, and the ARG7 3’ UTR were amplified in two pieces from a cDNA preparation from the Chlamydomonas reinhardtii strain CC-1010. These fragments were then fused into Hcr1 with the USER enzyme.

Ampicillin resistant in E. coli; restoration of arginine prototrophy in certain Chlamydomonas strains (tested in CC-1819, CC-1820, CC-1826).

Note: This promoter is not determined to be the minimal sequence for promoter activity. This cut-off was chosen because this is how much was included in a previous genomic fragment that was shown to not need an additional promoter. It is possible that this vector could be made even smaller by truncating some sequence at the 5’ end.

host strain:DH5α
amp resistant

Sequence (.gb file)

From Adrian Valli, David Baulcombe lab, University of Cambridge, UK, March 2016

This construct is able to express a nitrate-inducible artificial miRNA of interest. It confers resistance to ampicillin (carbenicillin) in E. coli, and resistance to paromomycin in transgenic Chlamydomonas lines. For further details, please see Valli et al., 2016, Genome Research. In this manuscript the plasmid is under the name of ni-amiRNA.

host strain: DH5α
amp resistant

Sequence

Valli AA, Santos BA, Hnatova S, Bassett AR, Molnar A, Chung BY, Baulcombe DC (2016) Most microRNAs in the single-cell alga Chlamydomonas reinhardtii are produced by Dicer-like 3-mediated cleavage of introns and untranslated regions of coding RNAs. Genome Res. 26:519-29

From Adrian Valli, David Baulcombe lab, University of Cambridge, UK, March 2016

This construct expresses a nitrate-inducible artificial miRNA against PSY. It confers resistance to ampicillin (carbenicillin) in E. coli, and resistance to paromomycin in transgenic Chlamydomonas lines. For further details, please see Valli et al., 2016, Genome Research. In this manuscript the plasmid is under the name of ni-amiRNA-PSY.

host strain: DH5α
amp resistant

Sequence

Valli AA, Santos BA, Hnatova S, Bassett AR, Molnar A, Chung BY, Baulcombe DC (2016) Most microRNAs in the single-cell alga Chlamydomonas reinhardtii are produced by Dicer-like 3-mediated cleavage of introns and untranslated regions of coding RNAs. Genome Res. 26:519-29

From Adrian Valli, David Baulcombe lab, University of Cambridge, UK, March 2016

This construct expresses the spectinomycin resistance gene (derived from pALM32) under a constitutive promoter. It confers resistance to ampicillin (carbenicillin) in E. coli and resistance to paromomycin in transgenic Chlamydomonas lines. This construct in particular carries an intron in the middle of the spectinomycin resistance gene that lacks the miRNA stem-loop. For further details, please see Valli et al., 2016, Genome Research. In this manuscript the plasmid is under the name of spect/intron.

host strain: DH5α
amp resistant

Sequence

Valli AA, Santos BA, Hnatova S, Bassett AR, Molnar A, Chung BY, Baulcombe DC (2016) Most microRNAs in the single-cell alga Chlamydomonas reinhardtii are produced by Dicer-like 3-mediated cleavage of introns and untranslated regions of coding RNAs. Genome Res. 26:519-29

From Adrian Valli, David Baulcombe lab, University of Cambridge, UK, March 2016

This construct expresses the spectinomycin resistance gene (derived from pALM32) under a constitutive promoter. This construct in particular carries an intron in the middle of spectinomycin resistance gene that contains a miRNA stem-loop, which produces an artificial miRNA against Maa7. For further details, please see Valli et al., 2016, Genome Research. In this manuscript the plasmid is under the name of spect/intron(mi).

host strain: DH5α
amp resistant

Sequence

Valli AA, Santos BA, Hnatova S, Bassett AR, Molnar A, Chung BY, Baulcombe DC (2016) Most microRNAs in the single-cell alga Chlamydomonas reinhardtii are produced by Dicer-like 3-mediated cleavage of introns and untranslated regions of coding RNAs. Genome Res. 26:519-29

From Rosanna Young and Saul Purton, University College London, UK, July 2016

pSRSapI is a vector for expressing transgenes in the C. reinhardtiii chloroplast (Wannathong et al., 2016). The transgene should be inserted into the expression site using SapI and SphI restriction enzymes (or their isoschizomers LguI and PaeI, respectively). It will then be flanked by the C. reinhardtii psaA exon 1 promoter and 5′ UTR (perfect scarless fusion at the ATG start codon) and the C. reinhardtii rbcL 3′ UTR. The external flanking regions in the construct include an intact psbH gene and target the expression cassette downstream of psbH by homologous recombination.

Ampicillin selection in E. coli. Minimal medium for restoration of psbH gene in C. reinhardtii, selecting for phototrophic growth. The recipient cell line must therefore be a psbH mutant such as CC-5168 cw15 ∆psbH [strain TN72].

Sequence

Wannathong T, Waterhouse J, Young R, Economou C, Purton S (2016) New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii. Applied Microbiology & Biotechnology 100 (12): 5467-77

Young R, Purton S (2016) Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnology Journal 14 (5): 1251-60

Young R, Purton S (2014) Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression. The Plant Journal 80 (5): 915-25

From Rosanna Young, Saul Purton lab, University College London, UK, March 2017

pWUCA1 can be used to introduce the trnWUCA gene downstream of psaA exon 3 in the C. reinhardtii chloroplast genome using spectinomycin selection. trnWUCA is a synthetic tRNA that allows the readthrough of internal UGA codons.

100 μg/ml ampicillin in E. coli. 100 μg/ml spectinomycin for selection of aadA cassette in C. reinhardtii.

Sequence

Young RE & Purton S (2016) Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnol J. 14:251-60

From Rosanna Young, Saul Purton lab, University College London, UK, March 2017

pWUCA2 can be used to introduce the trnWUCA gene downstream of psbH in the C. reinhardtii chloroplast genome. trnWUCA is a synthetic tRNA that allows the readthrough of internal UGA codons. This plasmid also contains an empty expression cassette, where a gene of interest can be inserted (e.g. one with internal TGA codons).

100 μg/ml ampicillin in E. coli. Minimal medium for selection of restoration of psbH gene in C. reinhardtii (a psbH mutant recipient line must be used, e.g. TN72)

Sequence

Young RE & Purton S (2016) Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnol J. 14:251-60

From Don Weeks, University of Nebraska, April 2017

pWEN1 is spectinomycin resistant in E. coli and has the complete DNA sequence of the Cas9/intron-sgRNA gene targeting the exogenous mutant GFP gene for Chlamy.

host strain: DH5α

Sequence (ApE file)
Algal Research Cas9-intron-sgRNA Manuscript
Algal Research Supplemental Data

Wen Zhi Jiang, Donald P. Weeks, A gene-within-a-gene Cas9/sgRNA hybrid construct enables gene editing and gene replacement strategies in Chlamydomonas reinhardtii, In Algal Research, Volume 26, 2017, Pages 474-480, ISSN 2211-9264
(http://www.sciencedirect.com/science/article/pii/S2211926417302138)

From Don Weeks, University of Nebraska, April 2017

pWEN2 is kanamycin resistant in E. coli and has the complete DNA sequence of the Cas9/intron-sgRNA gene targeting the native PDS3-1 gene.

host strain: DH5α

Sequence (ApE file)
Algal Research Cas9-intron-sgRNA Manuscript
Algal Research Supplemental Data

Wen Zhi Jiang, Donald P. Weeks, A gene-within-a-gene Cas9/sgRNA hybrid construct enables gene editing and gene replacement strategies in Chlamydomonas reinhardtii, In Algal Research, Volume 26, 2017, Pages 474-480, ISSN 2211-9264
(http://www.sciencedirect.com/science/article/pii/S2211926417302138)

From Don Weeks, University of Nebraska, April 2017

pWEN3 is kanamycin resistant in E. coli and has the complete DNA sequence of the Cas9/intron-sgRNA gene targeting the native PDS3-2 gene.

host strain: DH5α

Sequence (ApE file)
Algal Research Cas9-intron-sgRNA Manuscript
Algal Research Supplemental Data

Wen Zhi Jiang, Donald P. Weeks, A gene-within-a-gene Cas9/sgRNA hybrid construct enables gene editing and gene replacement strategies in Chlamydomonas reinhardtii, In Algal Research, Volume 26, 2017, Pages 474-480, ISSN 2211-9264
(http://www.sciencedirect.com/science/article/pii/S2211926417302138)

From Don Weeks, University of Nebraska, April 2017

pWEN4 is ampicillian resistant in E. coli and has the exogenous mutant Ble gene placed in a plasmid containing the cas9/intron-sgRNA gene.

host strain: DH5α

Sequence (ApE file)
Algal Research Cas9-intron-sgRNA Manuscript
Algal Research Supplemental Data

Wen Zhi Jiang, Donald P. Weeks, A gene-within-a-gene Cas9/sgRNA hybrid construct enables gene editing and gene replacement strategies in Chlamydomonas reinhardtii, In Algal Research, Volume 26, 2017, Pages 474-480, ISSN 2211-9264
(http://www.sciencedirect.com/science/article/pii/S2211926417302138)

From Don Weeks, University of Nebraska, April 2017

pWEN5 is ampicillian resistant in E. coli and has the complete DNA sequence of the mutant Ble gene containing a Cas9/intron-sgRNA target site identical to the target site in the KU70 gene in Chlamy.

host strain: DH5α

Sequence (ApE file)
Algal Research Cas9-intron-sgRNA Manuscript
Algal Research Supplemental Data

Wen Zhi Jiang, Donald P. Weeks, A gene-within-a-gene Cas9/sgRNA hybrid construct enables gene editing and gene replacement strategies in Chlamydomonas reinhardtii, In Algal Research, Volume 26, 2017, Pages 474-480, ISSN 2211-9264
(http://www.sciencedirect.com/science/article/pii/S2211926417302138)

From Don Weeks, University of Nebraska, April 2017

pWEN6 is ampicillian resistant in E. coli and has the complete DNA sequence of the Cas9/intron-sgRNA target site identical to the target site in the KU70 gene in Chlamy.

host strain: DH5α

Sequence (ApE file)
Algal Research Cas9-intron-sgRNA Manuscript
Algal Research Supplemental Data

Wen Zhi Jiang, Donald P. Weeks, A gene-within-a-gene Cas9/sgRNA hybrid construct enables gene editing and gene replacement strategies in Chlamydomonas reinhardtii, In Algal Research, Volume 26, 2017, Pages 474-480, ISSN 2211-9264
(http://www.sciencedirect.com/science/article/pii/S2211926417302138)