# Cyanobacteria as Chassis for Industrial Biotechnology: Progress and Prospects
## 3 Classical Genetic Engineering Technology
### Selectable Markers  
It is essential to be able to select for rare transformant events amongst a large population of treated cyanobacterial cells [4; 11]. Systematic and efficient methods whereby successful transformants can be identified and isolated are well established. Usually, host cells that contain the transferred DNA are identified by transferring a gene whose product has the potential to inactivate an antimetabolite, such as an antibiotic [22]. When cultured in the presence of the antimetabolite, only those cells to which the gene (normally an antibiotic resistance cassette) has been transferred should grow. Since cyanobacteria are Gram-negative bacteria, antibiotics and antibiotic-resistant genes that are known to be effective for that group of bacteria have been extensively exploited [11].  
Antibiotics used as selectable markers in cyanobacteria, in general, include neomycin and kanamycin and the corresponding neomycin phosphotransferase (npt)-encoding genes from transposons Tn5 and Tn903, and streptomycin and spectinomycin and the corresponding aminoglycosade adenyltransferase (aadA)-encoding genes from Tn7 [87] and the omega interposon [88]. Bleomycin, although expensive, is sufficiently active to be cost effective with _Anabaena_ 7120. Chloramphenicol and chloramphenicol acetyl transferase (cat) have proven useful for unicellular cyanobacteria [89] and _Anabaena_ sp. strain 90 [90]. Nourseotricine has been reported recently to be effective in a study on an RNA polymerase mutant of _Synechocystis_ 6803 [91]. Antibiotics such as tetracycline and rifampicin, which are light sensitive, are of much reduced usefulness for cyanobacteria [21; 22]. Other antibiotics with the same property include erythromycincin [10], zeocin [92]and gentamicin [93]. However, gentamicin and erythromycincin use in cyanobacteria induces oxidative damage and should be used with caution [94; 95].  
_Synechocystis_ 6803 specifically shows preference to some antibiotics. For example, it has been reported that the transformants grew 4-6 days earlier in the plates containing kanamycin than those containing chloromycetin [73]. Furthermore, the bacterial promoter and termination sequences of kanamycin is recognised by _Synechocystis_ 6803 and hence can be used without further modifications [11]. A summary of the selectable markers (antibiotic resistance markers) suitable for use in _Synechocystis_ 6803 can be found in (Table 2) while the same combinations of antibiotics can be tested with closely related species of cyanobacteria. By using a combination of antibiotic resistance cassettes, multiple genes can be inactivated in a single cyanobacterial strain [96; 97; 98]. However, it should be noted, when using multiple antibiotics resistance cassettes, that in some cases using one type of antibiotic resistance cassette may lead to resistance towards another. For example, the cassette normally used for spectinomycin resistance also results in streptomycin resistance. Therefore, it is necessary to introduce the streptomycin resistance cassette before the spectinomycin resistance cassette [11].