Part:BBa_K3930028

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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 590
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 777
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 590
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 590
  
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

This integrative locus NSI left arm of Synechococcus elongatus genome comes from the plasmid pAM4951 Taton et al. (2014). This part is flanking the insert in 5', and must be used with the integrative locus (BBa_K3930027) NSI right arm part in 3'.

Results

Integration of part (BBa_K3930026) into the cyanobacterium genome

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

Figure 1: Integration of pCONCOMBRE insert in the cyanobacterium genome

Insert integration was checked by PCR visualized on EtBr stained agarose electrophoresis gel. A theoretical gel is presented on the right (note that a different ladder is presented on the theoretical gel). Most expected sizes were obtained, but one amplicon for the left arm (LA) had not the right size. However, all genes from the insert were detected.

We concluded that the integrative locus NSI left arm (BBa_K3930028) coupled with the integrative locus (BBa_K3930027) NSI right arm part is nonetheless functional under these 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.