Part:BBa_K3520019

pHimarEm1+TUs of BcsA,B,C,D

Usage and Biology

The pMORPHÆ plasmid, consists of the pHimarEm1 vector (BBa_K3520009) containing the transcriptional units of the bcs operon’s genes (A. xylinous). More specifically, this composite vector was designed in order to permanently insert these genes into the Flavobacterium’s genome, as to produce cellulose. This plasmid was made by connecting the parts BBa_K3520013, BBa_K3520014, BBa_K3520015, BBa_K3520016, designed by iGEM Athens.

Plasmid map

Figure 1: pMorphae complete plasmid map with all TUs.

The present plasmid is used to insert the Bcs operon genes (X54676.1) to a bacterium of the Flavobacterium genus through conjugation using an Escherichia coli strain for conjugation. Each gene has its own transcriptional unit that will allow their constitutive expression. These genes will all intergrated into the genome of the target Flavobacterium with the help of the Mariner transposon and then the Cellulose is produced by the recombinant bacteria.

Type IIS assembly design

Figure 2: Type IIS for level 1 Golden Gate assembly with SapI.

Gene circuitry

Figure 3: The interactions regulating the production of bacterial cellulose under the bcs operon.

Athens 2020

The current part is designed by iGEM Athens 2020 team during the project MORPHÆ. In this project, Flavobacteria were used to produce a non-cellular structurally coloured biomaterial which would require the secretion of a biomolecule that Flavobacteria do not normally secrete. Our hypothesis is that the formed matrix will have a structure similar to that of the biofilm and thus, it will provide the material with macroscopically the same colouration properties as the biofilm.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 16429
  Illegal suffix found in sequence at 9767
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 16429
  Illegal NheI site found at 10096
  Illegal SpeI site found at 9768
  Illegal PstI site found at 9782
  Illegal NotI site found at 9775
  Illegal NotI site found at 16435
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 16429
  Illegal BglII site found at 10418
  Illegal BglII site found at 12957
  Illegal BglII site found at 13616
  Illegal XhoI site found at 10053
  Illegal XhoI site found at 13254
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 16429
  Illegal suffix found in sequence at 9768
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 16429
  Illegal XbaI site found at 16444
  Illegal SpeI site found at 9768
  Illegal PstI site found at 9782
  Illegal NgoMIV site found at 11082
  Illegal AgeI site found at 12649
  
• 1000
  COMPATIBLE WITH RFC[1000]