Revision as of 01:03, 2 October 2024 (view source) Emmazhou (Talk | contribs) ← Older edit		Revision as of 01:10, 2 October 2024 (view source) Emmazhou (Talk | contribs) Newer edit →
Line 2:		Line 2:
	__NOTOC__		__NOTOC__
	<partinfo>BBa_K5175006 short</partinfo>		<partinfo>BBa_K5175006 short</partinfo>
		+
	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.		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.
Line 45:		Line 46:
	width="700"		width="700"
	title="">		title="">
−	<figcaption>Fig.2  The bands of pTerephthalate-A and pTerephthalate-B from PCR<br><br>TThe 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.<br><br>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.	+	<figcaption>Fig.2  The bands of pTerephthalate-A and pTerephthalate-B from PCR<br><br>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.<br><br>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.
	</figcaption>		</figcaption>
	</figure>		</figure>

---

Revision as of 01:10, 2 October 2024

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.

Sequence and Features

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). TphB is a dehydrogenase that oxidizes the diol moiety (two hydroxyl groups) of DCD to a keto group, resulting in the production of PCA.TphA2, TphA3 constitute the large and small subunits of the TPADO oxidase component responsible for binding to the TPA substrate and catalyzing the oxygenation reaction in the active site.TphA2 contains the active site in direct contact with the substrate, TPA, and contains the Cys-X1-His X17-Cys-X2-His pattern, binds to Rieske-type [2Fe-2S] iron-sulfur clusters and participates in electron transfer, which is a key part of the catalytic reaction of dioxygenases , TphA3 participates in the construction of the substrate channel or the appropriate positioning of the active site, and assists TphA2 in completing the oxidation reaction; TphA1 does not directly participate in the oxygenation reaction, but it 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.

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 .