Part:BBa_K3930027

Integrative right site in the NSI locus of the S. elongatus genome Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 441
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 441
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 441
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 441
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 651
  

Introduction

Integrative locus NSI right arm of Synechococcus elongatus genome comes from the plasmid pAM4951 (Taton et al. 2014). This part needs to flank the insertion in 3', and must be used with the integrative locus (BBa_K3930028) NSI left arm part in 5'.

Results

Integration of the part (BBa_K3930026) into the cyanobacterium genome

The part (BBa_K3930026) was transformed into the S.elongatus UTEX 2973 according to the triparental conjugation protocol of Gale et al. (2019). The construction is flanked by the part (BBa_K3930027) and (BBa_K3930028). Figure 1 shows the electrophoresis gel of PCR on colony to verify clones.

Figure 1: Integration of pCONCOMBRE in the cyanobacterium genome

pCONCOMBRE was checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented on the right of each gel and the NEB 1 kb DNA ladder on the left (note that a different ladder is presented on the theoretical gel)

The Integrative locus NSI right arm (BBa_K3930027) coupled with the integrative locus (BBa_K3930028) NSI left arm part work under those experimental conditions.

References

1. Gale GAR, Osorio AAS, Puzorjov A, Wang B, McCormick AJ. 2019. Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit. JoVE (Journal of Visualized Experiments).(152):e60451. doi:10.3791/60451.
2. Taton A, Unglaub F, Wright NE, Zeng WY, Paz-Yepes J, Brahamsha B, Palenik B, Peterson TC, Haerizadeh F, Golden SS, et al. 2014. Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria. Nucleic Acids Res. 42(17):e136. doi:10.1093/nar/gku673.