Part:BBa_K4649010

PqqC + gcd codon optimized for Rhizobium tropici

The PqqC gene encodes pyrroloquinoline-quinone synthase, which is one part of the Pqq operon in A. radiobacter. It oxides Ahqq into pyrroloquinoline-quinone (Pqq). The gcd genes code for the enzyme glucose dehydrogenase, which oxidizes glucose into gluconic acid with the help of Pqq, acting as a cofactor. This part is important because it increases the solubilization of inorganic phosphate, making it available for plants that have a symbiotic relationship with our chosen chassis, rhizobium. We designed this part by codon optimizing it for rhizobia in Benchling. We obtained this DNA from IDT. We encountered an issue with the gcd sequence when we ordered them from IDT due to the high gc content in the gene sequence. This part can be used in an biosafety level laboratory. To test this part, we ran a pcr with primers specific to this part, and ran a gel. Since the gel yielded positive results, we know that this part was successfully expressed.

We ran an electrophoresis gel to test whether our genes were successfully amplified with PCR.    

We were able to see that the gene was successfully inserted into our pBBR1-MCS-2 plasmid and cloned first into E. coli and then into Rhizobium (see clones 3-3, 3-4, 4-2, 4-3, 4-4, 4-5, and 4-6):

We also expressed the protein by inducing it with IPTG and running an SDS-PAGE protein gel, and we could see a band of about 87kD, which is the size that we expect for the GDH protein:

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2100
  Illegal XbaI site found at 3328
  Illegal SpeI site found at 3322
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2100
  Illegal SpeI site found at 3322
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2100
  Illegal BamHI site found at 2049
  Illegal BamHI site found at 3316
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2100
  Illegal XbaI site found at 3328
  Illegal SpeI site found at 3322
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2100
  Illegal XbaI site found at 3328
  Illegal SpeI site found at 3322
  Illegal NgoMIV site found at 479
  Illegal NgoMIV site found at 1033
  Illegal NgoMIV site found at 1309
  Illegal NgoMIV site found at 1362
  Illegal NgoMIV site found at 1401
  Illegal NgoMIV site found at 2895
  Illegal NgoMIV site found at 3052
  
• 1000
  COMPATIBLE WITH RFC[1000]