# Cyanobacteria as Chassis for Industrial Biotechnology: Progress and Prospects
## 3 Classical Genetic Engineering Technology
### Plasmid Expression Vectors  
Plasmid vectors that are used in transformation of cyanobacteria can be classified into two main groups [22]. The first group is the non-replicating plasmids or integrative plasmids and these cannot replicate independently and would eventually be lost through successive cell divisions [26]. The other group is commonly referred to as replicative plasmids, shuttle vectors or biphasic plasmids and are capable of replication in both cyanobacteria and _E. coli_[22; 26; 37] as they most often contain replication sequences from both _E. coli_ and the cyanobacterial host [21]. Both integrative and replicative plasmids have been developed for cyanobacteria and have their experimental uses. For example, the ideal plasmid vector for transporting DNA that must either transpose (e.g., a transposon) or integrate (e.g., by homologous recombination) in order to be stably maintained are the integrative vectors [6; 86]. These vectors are ideal for targeted mutagenesis in which the gene in the chromosome is replaced by the mutated gene in the plasmid [6] or in which the cyanobacterial host needs to select for the recombination of the plasmid into the chromosome [76]. It has been observed that none of the common cloning vectors from _E. coli_ seem to be able to replicate in cyanobacteria [4], presumably because the colE1 origin of replication, carried on vectors such as pUC19, pBluescript and pGEM-T, are not recognised by the DNA-replication machinery of cyanobacteria [86].  
Replicative plasmids can be classified into two types: those that contain replicons of broad-host range plasmids [102; 103] and those derived from endogenous cryptic plasmids [37; 104]. Replicative plasmids carrying a variety of selectable markers have been constructed for cyanobacteria [6]. Examples include the pFCLV 7 [89] and plasmids derived from RSF101 (e.g., pSL211, pPMQAK1 and pFC1) [26; 29; 102]. Replicative plasmids that can replicate in multiple hosts (either broad or narrow host range) have the potential to express an exogenous gene in numerous hosts [22]. Nevertheless, most shuttle vectors for cyanobacteria are still not well characterised for their copy number [21], and  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Selectable Marker** & **Source** & **Concentration (\#g/mL)** & **References** \\ \hline Chloramphenicol & pBR325 & 5–150 & [9; 90] \\ Erythromycin & pRL425 & 5–300 & [9] \\ Kanamycin & pUC4K (Tn 903) & 5–500 & [9; 90] \\ Spectinomycin & pHP45\(\Omega\) & 3–250 & [6; 9] \\ Neomycin & Tn5 & NA & [32] \\ Streptomycin & Tn7 & NA & [99] \\ Spectinomycin & pH45\(\Omega\) (Tn7) & 25 & [90; 99] \\ Zeocin & - & 25 & [100] \\ Gentamicin & - & NA & [101] \\ \hline \hline \end{tabular} NA: not available.  
\end{table}
Table 2: Selectable markers (antibiotic resistance markers) suitable for use in _Synechocystis_.  
the lack of shuttle vectors with a varied copy number puts limitations on the controlled expression of heterologously expressed genes by selecting their copy number [99]. For example, the copy number from plasmids derived from RSF1010 are ten per chromosome in _E. coli_ cells and approximately 10-30 per cell in _Synechocystis_ 6803 (\(\sim\)1-3 per chromosome) [102; 103]. In a recent study, it was reported that the GT wild-type strain contains 142 genome copies in the exponential phase and 42 genome copies in the linear and stationary growth phase [105]. The copy number of other broad host-range plasmids have not been quantified to date [29], which adds a limitation to their use. A recent review on shuttle vectors used in various cyanobacterial hosts is presented in [26].  
In terms of transformation efficiency, it has been reported that transformation of _Synechocystis_ 6803 with plasmids obtained from _Synechocystis_ 6803 cultures result in 10-20 times better transformation rates than transformations done using bacterial plasmids, perhaps due to a compatible DNA methylation pattern. Nevertheless, plasmids extracted from _E. coli_ are routinely used as they are much more convenient to prepare [106].