Part:BBa_K3726028

CDS_Lv0_MCG-PK

This composite part corresponds to a phytobrick compatible MoClo Lv.0 CDS element. This part is an artificial operon designed to express the enzymes CDS_PtaBs (BBa_K3726019), CDS_PKPa (BBa_K3726021), CDS_mdh (BBa_K3726023), CDS_GarK (BBa_K3726025), garR_CDS (BBa_K3726027).

Within this artificial operon each CDS element is preceded by an in-silico designed RBS sequence using DeNovo DNA software. Only the first coding sequence of the operon is not preceded by an RBS sequence which must be assembled during the level 1 MoClo reaction for the expression of the operon in a level 1 transcriptional unit.

This part has been used within the iGEM MADRID_UCM 2021 iGEM team for the expression of an artificial carbon fixation pathway & bypass towards acetyl-CoA formation.

Within this polycistronic CDS part, the first two enzymes correspond with a phosphoketolase bypass (PKpa-PtaBs), while the remaining corresponds with the first half of an artificial pathway designed to enhance acetyl-CoA pool and enhance carbon fixation.

The relative transcription initiation strengths of each RBS in the operon context has been calculated with DeNovo DNA Software. Results are depicted in the graphic below.

For PK-MCG

Since this part corresponds with the half part of a fully functional artificial carbon fixation pathway & bypass towards acetyl-CoA formation, relative expression of each enzyme required for optimal pathway functioning is controlled by their internal RBS.

As can be observed, the PtaBs initial RBS sequence have been designed to pose a moderate strength. It has been designed this way because there is another native PtaBs variant within the PCC 11801 genome. Nevertheless, PtaBs which come from bacillus subtilis, has higher specific activity levels than the native one. Also, it has been designed to pose a moderate strength because PtaBs is the first enzyme of the operon and so on it expresses itself higher than the others.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 3079
  Illegal PstI site found at 3708
  Illegal PstI site found at 4205
  Illegal PstI site found at 4804
  Illegal PstI site found at 5742
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 3079
  Illegal PstI site found at 3708
  Illegal PstI site found at 4205
  Illegal PstI site found at 4804
  Illegal PstI site found at 5742
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1618
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 3079
  Illegal PstI site found at 3708
  Illegal PstI site found at 4205
  Illegal PstI site found at 4804
  Illegal PstI site found at 5742
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 3079
  Illegal PstI site found at 3708
  Illegal PstI site found at 4205
  Illegal PstI site found at 4804
  Illegal PstI site found at 5742
  Illegal NgoMIV site found at 1195
  Illegal NgoMIV site found at 3304
  Illegal NgoMIV site found at 3764
  Illegal AgeI site found at 5317
  Illegal AgeI site found at 5400
  
• 1000
  COMPATIBLE WITH RFC[1000]