Part:BBa_K3971028

Cassette to insert sps gene in the Neutral Site 3 of the S. elongatus UTEX 2973 genome.

This composite part has been designed to insert the sps gene into the Neutral Site 3 of the S. elongatus UTEX 2973 genome.

The promoter in this part is PcpcB-m6 (BBa_K3971000). In order to allow for modularity in the promoter region of the cassette, two restriction sites Nde1 and BamH1 flank it, so that the promoter can be easily excised out and replaced with another. Thus one can study the effect of different promoters on sucrose production efficiency.

Both ends of the composite part contain Kpn1 restriction sites since this cassette is designed to be cloned into the pSL2680 plasmid, which is a Cpf1 based CRISPR plasmid [1], which has a Kpn1 site and a Sal1 site to allow for cloning of cassettes.

The sps gene is native to the Synechococcus sp, and the sps catalyzed reaction is the rate limiting step in sucrose biosynthesis in cyanobacteria [1]. It catalyzes the rate-limiting conversion of uridine diphosphate glucose (UDP-Glu) and fructose 6-phosphate (F6P) into sucrose 6-phosphate (S6P) in the sucrose synthesis pathway.

We aimed to overexpress the sps gene in order to increase the amount of sucrose produced by S. elongatus when exposed to salt stress.

Figure of the composite construct for inserting the sps gene into the Neutral Site 3 of the S. elongatus UTEX 2973 genome. NS3a and NS3b are the two homology arms to Neutral Site 3.

References

1: Du, Wei, et al. "Exploring the photosynthetic production capacity of sucrose by cyanobacteria." Metabolic engineering 19 (2013): 17-25.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2877
  Illegal PstI site found at 644
  Illegal PstI site found at 2915
  Illegal PstI site found at 3636
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2877
  Illegal NheI site found at 3070
  Illegal PstI site found at 644
  Illegal PstI site found at 2915
  Illegal PstI site found at 3636
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2877
  Illegal BamHI site found at 1196
  Illegal XhoI site found at 2654
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2877
  Illegal PstI site found at 644
  Illegal PstI site found at 2915
  Illegal PstI site found at 3636
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2877
  Illegal PstI site found at 644
  Illegal PstI site found at 2915
  Illegal PstI site found at 3636
  
• 1000
  COMPATIBLE WITH RFC[1000]