A prototypic TTT consists of a periplasmic substrate binding protein and two integral membrane proteins (Antoine <em>et al</em>. 2005).
In the case of the TPA uptake system, TphC corresponds to the SBP (that binds strictly to TPA) and TpiA-TpiB to the integral membrane components (Hosaka <em>et al</em>. 2013, Gautom <em>et al</em>. 2021).
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In the study by Hosaka <em>et al</em>. (2013), coexpression of the TPA transporter and the tph genes encoding TPADO and DCDDH in <em>p. putida</em> Pp Y1100, showed that tpiBA (encoding for an operon containing both TpiA and TpiB) and tphC both were necessary for the resting cells' conversion of TPA.
In the study by Hosaka <em>et al</em>. (2013), coexpression of the TPA transporter and the tph genes encoding TPADO and DCDDH in <em>Pseudomonas putida</em> Pp Y1100, showed that tpiBA (encoding for an operon containing both TpiA and TpiB) and tphC both were necessary for the resting cells' conversion of TPA.
The TpiA-TpiB membrane components were also strongly suggested to interact with other molecules than TPA since disruption of the genes also caused growth deficiency of <em>Comamonas</em> sp. strain E6 on IPA, OPA, PCA and citrate.
Additionally, TPA uptake seemed to be inhibited by the presence of PCA as well as complete inhibition by the protonophore CCCP.
The latter observation made by the authors indicates that the system is dependent on proton motive force.
<p>Plasmid pJCBAtG (Figure 1) was kindly provided by the authors behind Hosaka <em>et al</em>. 2013, at the Department of Bioengineering at Nagaoka University of Technology in Japan.
pJCBAtG contains the genes for the TPA uptake system (tpiA, tpiB and tphC), TPADO complex (tphA1, tphA2, tphA3) and DCDDH (tphB) under P<ub>m</ub> promoter control.
pJCBAtG contains the genes for the TPA uptake system (tpiA, tpiB and tphC), TPADO complex (tphA1, tphA2, tphA3) and DCDDH (tphB) under P<sub>m</sub> promoter control.
The plasmid also contains tetracycline (Tet) resistance and was constructed for use in <em>P. putida</em>.
The use of the P<sub>m</sub> promoter control system in <em>E. coli</em> has been shown to in general generate much less transcripts than with the T7 promoter (Balzer <em>et al</em>. 2013).
However, the P<sub>m</sub> system has been shown to be capable of expressing high levels of proteins in high cell density cultivations (Sletta <em>et al</em>. 2004).
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<imgstyle="width: 60%;"src='https://static.igem.wiki/teams/4378/wiki/img/lab-enzymes/tpa-transporter/tpa-transporter-dh5a.png'alt="Plate and restreaked plate of DH5α with TPA transporter">
<p><em>Figure2: (A) Agar plate containing tetracycline with DH5α cells transformed with the pJCBAtG plasmid.
One single colony grew in the overnight incubation at 37 <up>o</up>C, and a part of the colony had, prior to the picture being taken, been used for a restreak.
<p><em>Figure2: (A) Agar plate containing tetracycline with DH5α cells transformed with the pJCBAtG plasmid.
One single colony grew in the overnight incubation at 37 <sup>o</sup>C, and a part of the colony had, prior to the picture being taken, been used for a restreak.
(B) The restreaked plate from A, after overnight incubation at 37 <sup>o</sup>C, four days after the transformation.
<p>Attempt for protein overexpression was performed to investigate if TPADO (see “link to TPADO”) and TPA transporter was overexpressed in <em>E. coli</em> BL21.
<p>Attempt for protein overexpression was performed to investigate if TPADO (see <ahref="{{ url_for('pages', page='TPADO') }}">TPADO</a>) and TPA transporter was overexpressed in <em>E. coli</em> BL21.
No positive control was available for the P<sub>m</sub> promoter system, therefore only a negative (uninduced BL21 with pJCBAtG) control was used.
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The general protocol for protein overexpression was followed, with some modifications.
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Induction was done in 1:100 I and 1:10 I (hereinafter OD 0.8 I and OD 0.9 I) and no inducer was added to the (uninduced) controls (1:100 U and 1:10 U, hereinafter OD 0.8 U and OD 1 U respectively).
All mixtures were incubated for 10h at the desired conditions, after which they were transferred to a fridge.
11h later, cells were harvested by centrifuging at 4 <sup>o</sup>C and 4000 rpm for 5 min, resuspended in 2 mL buffer A and lysis of the resuspended cells was done with the SDS lysis protocol.
SDS-PAGE analysis was done with 7 𝝻L of the four samples' supernatants, and stained by the quick version.
SDS-PAGE analysis was done with 7 μL of the four samples' supernatants, and stained by the quick version.
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The SDS-PAGE analysis of the experiment showed no evidence for overexpression (see Figure 4) and the expression results were therefore inconclusive.
