Part:BBa_K1031620

NahF-Terminator

For detailed information concerning NahF, please visit 2013 Peking iGEM Adaptors

Introduction

NahF is a 50.8 KDa protein functioning as salicylaldehyde dehydrogenase to transform salicylaldehyde into salicylic acid (salicylate) using NAD⁺ (Fig.1). It is encoded in the naphthalene degradation plasmid from Pseudomonas putida, in which the oxidation of naphthalene has been extensively investigated. Plasmid pDTG1, NAH7 and pND6-1 identified in different P. putida strains all act to degrade naphthalene and share high identity in amino acid sequences <a href="#REF">^[11]</a>.

Figure.1 Biochemical reaction catalyzed by enzyme NahF
Salicylaldehyde is transformed into salicylic acid (salicylate) accompanied by the reduction of NAD⁺ to NADH.

NahF from plasmid NAH7 is most widely studied. It has a wide range of substrates (including salicylaldehyde, 5-chloro-salicylaldehyde, 3-nitro-benzaldehyde, 2-methoxy-benzaldehyde etc.) and its activity can be further enhanced 40.3% in the presence of Fe^{2+ [2]}. The wide range of substrates makes it an appropriate candidate to be an Adaptor since many aldehydes can be transformed to the corresponding acids that can be detected by NahR (for salicylates) or XylS (for benzoates).

NahF has been expressed in E.coli and its ability to catalyze the reaction in vitro and in vivo has been verified ^[3-4]. However, its reaction efficiency when expressed in E.coli was only about 3% of that when expressd in P. putida, possibly due to the difference of regulation in these two bacteria ^[3]. Therefore, it is necessary to fine-tune the expression level of NahF in E.coli. We built a library of constitutive promoters for tuning the expression of NahF, and NahR biosensor was used to detect the possible salicylates transformed from salicylaldehydes (Fig. 8).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 845
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1083
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 981
  Illegal BsaI.rc site found at 1135
  

Characterization of Biosensor

We built a library of constitutive promoters for tuning the expression of NahF, and NahR(BBa_K1031610) biosensor was used to detect the possible salicylates transformed from salicylaldehydes (Fig.2).

Fig.2 Schematic diagrams for the plasmid circuits used as Adaptor, NahF and the Sensor, NahR.
A constitutive promoter library for the expression of NahF was constructed to obtain the most appropriate expression level of NahF enzyme in E.coli. The number of the Standard Biological constitutive promoter Parts used in this study and its initiation strength is listed in the left portion of the figure. Promoters are presented in orange, RBS in light green, coding sequence in dark cyan, and terminators in dark red.

The performance of NahF Adaptor was tested following protocol similar to that of XylC. Bacteria carrying NahF enzyme was cultured overnight in LB medium at 37 °C and then diluted 100 fold into Minimal M9 medium, growed for 12 hours at 30 °C to transform salicylaldehyde into salicylate. Then the culture was centrifuged, supernatant was taken out to further culture NahR biosensor. Induction ratio was then measured by Flow Cytometry followed by Test Protocol 1.

The comparison of induction ratio (calculated as previously mentioned) of adaptor-equipped and unequipped NahR, showed that NahF highly improved the induction performance of biosensor NahR induced by salicylaldehyde and 5-chloro-salicylaldehyde (Fig.3a). Dose response curves for salicylaldehydes of NahR equipped with Adaptor NahF were also obtained (Fig.3b). Comparing it with the dose-response curve for salicylaldehydes of NahR following the same test protocol (Fig.3b,c), we demonstrated that Adaptor NahF also functioned to lower the detection limit (the concentration of inducer at which an output three times the basal single is generated) of salicylaldehyde (from 300 μM to 1 μM) (Fig.3b) and 5-chloro-salicylaldehyde (from 300 μM to 3 μM) (Fig. 3c). NahR's dose response curves for corresponding acids processed by NahF were obtained as well, showing that adding Adaptor NahF does not significantly influence the original characteristics of the biosensor. (Fig.3d).

Fig.3 Data plots that demonstrate the performance of Apator NahF.
(a) Induction ratio of biosensor NahR and NahR equipped with Adaptor NahF elicited by salicylaldehyde at the concentration of 1 mM and 5-chloro-salicylaldehyde at the concentration of 0.1 mM. Biosensor NahR with Adaptor NahF showed higher induction ratio than NahR. (b) Dose-response curves of Biosensor NahR and NahR equipped with NahF for salicylaldehyde. Use of Adaptor NahF significantly reduced the detection limit by more than 100 folds. (c) Dose-response curves of Biosensor NahR and NahR equipped with NahF for 5-chloro-salicylaldehyde. (d) Dose-response curves of Biosensor NahR and NahR equipped with NahF for salicylate and 5-chloro-salicylate. The almost overlapping curves for the two compounds showed that the Adaptor NahF did not interfere the performance of NahR biosensor.

In summary, Adaptors XylC and NahF functioned not only to optimize existing biosensors' response to several originally detectable compounds, but also expand detection profile in a new way besides modification on coding sequence such as mutagenesis or DNA shuffling. The advantages of the new concept that using adaptor to expand detection profile lie in that adding enzymes does not influence the original characteristics of the biosensors they adapt to, and this methodology does not require labor-intensive mutant screening work.

Reference

[1] Baoli Cai et. al, (2004) Complete nucleotide sequence and organization of the naphthalene catabolic plasmid pND6-1 from Pseudomonas sp. strain ND6, GENE, 336:231–240.

[2] Zhao H, Li Y, Chen W, Cai B. (2007) A novel salicylaldehyde dehydrogenase-NahV involved in catabolism of naphthalene from Pseudomonas putida ND6. Chin Sci Bull. 52(14):1942—8.

[3] M. A. Schell, (1983) Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7. J. Bacteriol. 153(2):822-829.

[4] R. W. Eaton and P. J. Chapman, (1992) Bacterial metabolism of naphthalene_construction and use of recombinant bacteria to study ring cleavage of 1,2-dihydroxynaphthalene and subsequent reactions. J. Bacteriol. 174(23):7542-7554.