Journal: Plant Physiology

Article Title: CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax ^{1 [OPEN]}

doi: 10.1104/pp.18.01401

Figure Lengend Snippet: Adaptation of the Plant Golden Gate MoClo level 0 syntax for generating level 1 assemblies for transfer to level T. A, The format for a level 0 MoClo acceptor vector with the part bordered by two BsaI sites. B, Typical level 0 parts from the Plant MoClo kit (Engler et al., 2014), where parts of the same type are bordered by the same pair of fusion sites (for each fusion site, only the sequence of the top strand is shown). Note that the parts are not drawn to scale. C and D, The syntax of the Plant MoClo kit was adapted to generate level 0 parts for engineering marked and unmarked cyanobacterial mutant strains (Lea-Smith et al., 2016). E to I, To generate knock-in mutants, short linker parts (30 bp) were constructed to allow assembly of individual flanking sequences, or marker cassettes (AbR or sacB), in level 1 vectors for subsequent assembly in level T. J and K, Parts required for generating synthetic srRNA or CRISPRi level 1 constructs. See Supplemental Information S2 and S3 for workflows. 3U+Ter, 3′UTR and terminator; AbR, antibiotic resistance cassette; AbR DOWN LINKER, short sequence (∼30 bp) to provide CGCT overhang; AbR UP LINKER, short sequence (∼30 bp) to provide GAGG overhang; CDS2(stop), coding sequence with a stop codon; DOWN FLANK, flanking sequence downstream of target site; DOWN FLANK LINKER, short sequence (∼30 bp) to provide GGAG overhang; Prom+5U, promoter and 5′ UTR; Prom TSS, promoter transcription start site; sacB, levansucrase expression cassette; sacB UP LINKER, short sequence (∼30 bp) to provide GAGG overhang; sgRNA, single guide RNA; SP, signal peptide; srRNA, small regulatory RNA; UP FLANK, flanking sequence upstream of target site; UP FLANK LINKER, short sequence (∼30 bp) to provide CGCT overhang; UNMARK LINKER, short sequence to bridge UP FLANK and DOWN FLANK.

Article Snippet: The CyanoGate system integrates with the two-part Golden Gate MoClo Plant Tool Kit, which can be acquired from Addgene (standardized parts [Kit no. 1000000047] and backbone acceptor vectors [Kit no. 1000000044]; www.addgene.org ; Engler et al., 2014 ).

Techniques: Plasmid Preparation, Sequencing, Mutagenesis, Knock-In, Construct, Marker, Expressing

Journal: Plant Physiology

Article Title: CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax ^{1 [OPEN]}

doi: 10.1104/pp.18.01401

Figure Lengend Snippet: Extension of the Plant Golden Gate MoClo Assembly Standard for cyanobacterial transformation. Assembly relies on one of two Type IIS restriction endonuclease enzymes (BsaI or BpiI). Domesticated level 0 parts are assembled into level 1 vectors. Up to seven level 1 modules can be assembled directly into a level T cyanobacterial transformation vector, which consists of two subtypes (either a replicative or an integrative vector). Alternatively, larger vectors with more modules can be built by assembling level 1 modules into level M, then cycling assembly between level M and level P, and finally transferring from level P to level T. Antibiotic selection markers are shown for each level. Level T vectors are supplied with internal antibiotic selection markers (shown), but additional selection markers could be included from level 1 modules as required. See Supplemental Table S1 and Supplemental Information S4 for the full list and maps of level T acceptor vectors.

Article Snippet: The CyanoGate system integrates with the two-part Golden Gate MoClo Plant Tool Kit, which can be acquired from Addgene (standardized parts [Kit no. 1000000047] and backbone acceptor vectors [Kit no. 1000000044]; www.addgene.org ; Engler et al., 2014 ).

Techniques: Transformation Assay, Plasmid Preparation, Transferring, Selection