Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Abstract

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Development of a Biotechnology Platform for the Fast-Growing Cyanobacterium Synechococcus sp. PCC 11901

“…Assembly of the plasmid for generating marked mutants was performed using a standard Golden Gate one-pot digestion/ligation reaction [ 22 ]. The codA gene in each cassette is under control of the promoter J23101 [ 8 ].…”

Pilot scale production, extraction and purification of a thermostable phycocyanin from Synechocystis sp. PCC 6803

“…The expression cassette was assembled using the CyanoGate MoClo toolkit and consisted of the TeBACD operon driven by the native Synechocystis PC promoter (P cpc560; part pC0.005) and terminator (T cpc , part pC0.078) ( Puzorjov et al, 2021 , Vasudevan et al, 2019 ). Cultures were screened for the presence of the CT.353 TeBACD by PCR as described in Gale et al (2019) . The oligonucleotides used for amplification are described in the supplementary materials .…”

Production of thermostable phycocyanin in a mesophilic cyanobacterium

“…Variants of the PC operon were PCR amplified from the wild-type Synechocystis and T. elongatus genomic DNA using Q5 High-Fidelity DNA Polymerase (New England Biolabs) and assembled into Level 0 CDS acceptor vector (pICH41308). Parts were domesticated ( Bsa I and Bpi I recognition sites removed) as necessary using a method described previously ( Gale et al, 2019 ). Native PC operon promoter (P cpc560 , pC0.005) and terminator (T cpc , pC0.078) from Synechocystis were cloned upstream and downstream of all PC operon variants into Level 1 Position 2 (reverse) acceptor vector (pICH47811) ( Vasudevan et al, 2019 ).…”

“…Native PC operon promoter (P cpc560 , pC0.005) and terminator (T cpc , pC0.078) from Synechocystis were cloned upstream and downstream of all PC operon variants into Level 1 Position 2 (reverse) acceptor vector (pICH47811) ( Vasudevan et al, 2019 ). All Level 1 PC operon variants were assembled into the self-replicative pPMQAK1-T (pCAT.000) acceptor vector using a Level 1 Position 1 “Dummy” (pICH54011) and End-Link 2 (pICH50881) parts ( Gale et al, 2019 ; Vasudevan et al, 2019 ). All expression vectors were conjugated into an unmarked (i.e.…”

“…All expression vectors were conjugated into an unmarked (i.e. lacking an antibiotic selection cassette) Synechocystis ‘Olive’ mutant (Δ cpcBAC2C1 D ) (SynO) via triparental mating, as described in Gale et al (2019) .…”