T7 promoter- tphA2-tphA3 -tphA1-T7 terminator- T7 promoter - tphB-tpaK-T7 terminator

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 912
Illegal PstI site found at 949
Illegal PstI site found at 982
Illegal PstI site found at 1951
Illegal PstI site found at 4853
Illegal PstI site found at 5142
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 2826
Illegal NheI site found at 2891
Illegal NheI site found at 2947
Illegal NheI site found at 5409
Illegal PstI site found at 949
Illegal PstI site found at 982
Illegal PstI site found at 1951
Illegal PstI site found at 4853
Illegal PstI site found at 5142
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 426
Illegal BglII site found at 3189
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 912
Illegal PstI site found at 949
Illegal PstI site found at 982
Illegal PstI site found at 1951
Illegal PstI site found at 4853
Illegal PstI site found at 5142
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 912
Illegal PstI site found at 949
Illegal PstI site found at 982
Illegal PstI site found at 1951
Illegal PstI site found at 4853
Illegal PstI site found at 5142
Illegal NgoMIV site found at 477
Illegal NgoMIV site found at 1516
Illegal NgoMIV site found at 2266
Illegal NgoMIV site found at 3236
Illegal NgoMIV site found at 3911
1000
COMPATIBLE WITH RFC[1000]

Description

It is a composite component consisting of the T7 promoter, T7 terminator, target genes tphA2, tphA3, tphA1, tphB, tpaK. It is responsible for converting TPA to 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD), oxidising the diol moiety (two hydroxyl groups) of DCD to a keto group to result in the production of PCA and transporting TPA.

Usage and Biology

TPA 1,2-dioxygenase (TPADO) is a two-component oxygenase consisting of three parts, TphA1, TphA2, and TphA3, which together enable TPADO to effectively catalyze the oxidative reaction of TPA, converting TPA to the intermediate product 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD).

TphA2

TphA2 constitute the large subunits of the TPADO oxidase component responsible for binding to the TPA substrate and catalyzing the oxygenation reaction in the active site.

TphA3

TphA3 constitutes the small subunits of the TPADO oxidase component responsible for binding to the TPA substrate and catalyzing the oxygenation reaction in the active site.

TphA1

TphA1 contains a [2Fe -2S] iron-sulfur cluster and a flavin adenine dinucleotide (FAD) binding site that transfers electrons from an electron donor (e.g., NADPH) to the oxidized component of TPADO.

TphB

TphB is a dehydrogenase that oxidizes the diol moiety (two hydroxyl groups) of DCD to a keto group, resulting in the production of PCA.

Fig 1. tph gene cluster core mechanism of converting TPA to PCA

TpaK

tpaK is a TPA transporter protein does not require other proteins for TPA transportation

Molecular cloning

Initially, we transformed the company-synthesized plasmids containing designed sequences into E. coli DH5α for amplification, allowing us to obtain a sufficient quantity of plasmid DNA for subsequent experiments. Following this, colony PCR was performed to confirm successful transformation, and the required plasmids were subsequently extracted for further experimentation.Subsequently, we employed PCR to obtain the target fragments, which were then integrated into the requisite plasmids for our study.

We constructed three plasmids for P. putida KT2440: pTerephthalate-A, pTerephthalate-B, and pRhamnolipid. We verified the size of each plasmid as well as all the fragments involved in constructing the plasmids . The plasmids were successfully introduced into P. putida through electroporation. Given that our wild-type P. putida exhibits resistance to chloramphenicol, the plasmids incorporated a kanamycin resistance marker. Consequently, we employed dual antibiotic selection plates to effectively screen for successfully transformed engineered strains.

Fig.3 The bands of pTerephthalate-A and pTerephthalate-B from PCR

The bands of pTerephthalate-A（~3000bp）from PCR are identical to the theoretical lengths of 2959 bp, 2449 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.

The bands of pTerephthalate-B（~4000 bp、~2000 bp）from PCR are identical to the theoretical lengths of 4111 bp，2000 bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.

Expressing experiment

We cultivated engineered P. putida KT2440 strains, including (pTerephthalate-A), (pTerephthalate-B), and the empty vector (pBBR1-CS2), in M9 medium with 2 g/L terephthalic acid (TPA) as the sole carbon source. Given that terephthalic acid is present at a low concentration and is considered a non-conventional carbon source, we initially inoculated P. putida into 10 mL of LB medium for 48 hours. Following this incubation, the cells were collected via low-speed centrifugation and then resuspended in M9 medium. The optical density at 600 nm (OD600) was measured every 12 hours to monitor growth, and the methodology for obtaining the growth curves is described as follows.

Part:BBa_K5175037