Part:BBa_K4192121

Plac-obcA-obcB-Plac-Fpoar, Oxalate secretion gene circuit

The expression products of obcA and obcB are enzymes needed to produce oxalate from citric acid and acetyl-CoA. And FpOAR protein is able to efflux oxalate. The composite part of these three genes can efflux oxalic acid out of the bacteria. We have already tested it in Pseudomonas fluorescens.

Contribution From CAU_China 2022

Characterization

CAU_China 2022 used this part as their oxalate secretion circuit. We transfered this circuit into pUC18 and pBBR1MCS2 to adapt to different chassis bacteria. This phenotype, in the way we test it, is reflected in the presence of more reducing substance outside the bacteria or changes in pH.

In P. fluorescens 2P24, we used the oxalic acid content detection kit (BoxBio, Beijing), which measure oxalic acid by salicylic acid complexed iron colorimetric method, allowing us to conduct colorimetric test for oxalic acid and other reducing substances in parallel treated samples with the same OD.

We added the same amount of induced bacterial solution to each of the medium, and they were first adjusted to the same OD₅₉₅ with ddH₂O. The control group is P. fluorescens 2P24 with an empty pBBR1MCS2 vector. After shaking culture at 28 ℃ for 12 hours, they were induced by IPTG. After another 8 hours, the bacterial solution was collected before we used a vacuum concentrator concentrate the products to ten times theirs original concentration.

Fig.2 Standard curves of oxalate concentration and absorbance at 520 nm.

Fig.3 Oxalic acid concentration test using 96-well plate. Lighter the red, higher the oxalic acid concentration in the well.

Fig.4 Result of oxalic acid concentration in P. fluorescens

Control: empty vector, 2nd: BBa_K4192120, 3rd:BBa_K4192121. In: Bacteria lysate, detected the oxalic acid concentration inside of the bacteria, out: the supernate of bacteria solution ,detected the oxalic acid concentration outside of the bacteria.

According to the results of ANOVA (Duncan's method), between these groups have a significant impact on the experimental results. We can come to the conclusion that the added Plac improved the efflux efficiency of oxalic acid, and this is our resulting oxalic acid producing bacteria.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2735
  Illegal PstI site found at 397
  Illegal PstI site found at 2878
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2735
  Illegal PstI site found at 397
  Illegal PstI site found at 2878
  Illegal NotI site found at 2330
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2735
  Illegal BglII site found at 1541
  Illegal BglII site found at 3738
  Illegal XhoI site found at 304
  Illegal XhoI site found at 1428
  Illegal XhoI site found at 3550
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2735
  Illegal PstI site found at 397
  Illegal PstI site found at 2878
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2735
  Illegal PstI site found at 397
  Illegal PstI site found at 2878
  Illegal NgoMIV site found at 389
  Illegal NgoMIV site found at 720
  Illegal NgoMIV site found at 748
  Illegal NgoMIV site found at 784
  Illegal NgoMIV site found at 1014
  Illegal NgoMIV site found at 1791
  Illegal NgoMIV site found at 2208
  Illegal AgeI site found at 326
  
• 1000
  COMPATIBLE WITH RFC[1000]