Part:BBa_K5335034
A cluster of 2 genes that generate and transport TAA to kill nematodes
This circuit constitutively expresses cis-aconitate isomerase, TbrA, and trans-aconitate transporter, TbrB, under promoter 43. These two proteins enable the engineered bacteria to secrete TAA into the extracellular space and in our project, TAA serves as a nematode-killing small organic molecule.
Usage and Biology
TbrA is an aconite isomerase from Bacillus thuringiensis YBT-1520, which can catalyze the isomerization of cis-aconite to trans-aconite and its reverse reaction at the same time, but has a higher activity to catalyze the isomerization of cis-aconite to trans-aconite. TbrB is a transaconite transporter derived from Bacillus thuringiensis, which has the ability to transport TAA from the interior of the cell to the exterior. In Bacillus thuringiensis, it is located within the same gene cluster as TbrA. Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 1793
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 1793
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1366
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 1793
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 1793
Illegal AgeI site found at 1569 - 1000COMPATIBLE WITH RFC[1000]
Functional Verification Experiment
To detect the TAA producing of the circuit, the vector is transferred into Bacillus subtilis. In order to verify the ability of engineered bacteria to secrete TAA, we fermented 200 mL of engineered bacteria culture medium for 24 h and 32 h, respectively, and crude TAA was extracted from the supernatant and tested by HPLC. The results are as followed Figure 1..
Figure 1. The structure of expression vector. The plasmids is based on pHT254, inserting the circuit through homologous recombination.
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