Part:BBa_K2042006

Cyclohexanone inducible promoter

This is a sequence for a cyclohexanone inductible promoter. This promoter specially adapted for the use in gram negative bacteria [1], and Pseudomonas putida is a good candidate because it is able to thrive in environments containing haloalkanes and other cyclic carbon compounds[2]. It has been extracted from a SEVA (Standard European Vector Architecture) plasmid (Silvia-Rocha et al., 2013).

This promoter is based on elements of the cyclohexanol biodegradation pathway of Acinetobacter johnsonii edited in silico to obtain a standardized SEVA (Standard European Vector Architecture) expression cargo [3], and assembled to conceive the pSEVA2311 plasmid. It was edited and formatted from ChnR/PchnB regulatory node of Acinetobacter johnsonii to ease the targeted engineering of ectopic gene expression in Gram-negative bacteria. The system was assembled in a minimal DNA segment adopting a Synthetic Biology standard.

Transcriptional activation of the ChnR/PchnB expression system upon addition of cyclohexanone was evaluated in a wild-type Escherichia coli strain. The expression data presented reflect a tightly controlled transcription initiation signal in response to cyclohexanone. Key features of this expression vector include a very low expression level in the absence of inducer, high transcriptional capacity, an induction kinetics very similar in both minimal and rich culture media, and linear accumulation of the reporter product along time.

Informations on Cyclohexanone and its manipulation

Cyclohexanone is an organic compound which chemical formula is (CH2)5CO. It is a 6-carbon cyclic molecule that has a ketone functional group. In literature, it is reported that cyclohexanone exhibits selective toxic action on Pseudomonas putida with a toxicity threshold value of 180 mg/l. [5] However, as mentioned, concentrations of inducer (i.e. cyclohexanone) of 1 mM is suitable for gene expression in gram negative bacteria [1]

When working with cyclohexanone, we have to take into account it has effects in some lab materials. The most rellevant ones are immediat damages to polystyrene (PS) and some effect after 7 days of exposure to polypropilene (PP). Working on the lab, tips are usually made of PP. Its use for transient manipulation is not a problem. Other materials such as Eppendorfs tubes are also usually made of PP and it should be fine for protocol manipulation, but for longer storage purposes, alternative materials should be used. Chemical Compatibility Guide (Thermo Fisher Scientific)[6] contains a list of compounds where there are indicated compatibilities of CH and types of plastic materials.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

[1] Benedetti, Ilaria, Pablo I. Nikel, and Víctor de Lorenzo. "Data on the standardization of a cyclohexanone-responsive expression system for Gram-negative bacteria." Data in brief 6 (2016): 738-744.

[2] Benedetti, Ilaria, Víctor de Lorenzo, and Pablo I. Nikel. "Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes." Metabolic engineering 33 (2016): 109-118.

[3] Kim, Se Hyeuk, et al. "SEVA linkers: a versatile and automatable DNA backbone exchange standard for synthetic biology." ACS synthetic biology (2016).

[4] Silva-Rocha, E. et al. The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res. 41, D666–D675 (2013)

[5] Bringmann G, Kuehn R. WATRAG. Water Res 14 (3): 231-41 (1980)

[6] Chemical Compatibility Guide, from Thermo Fisher Scientific. Available at: http://sevierlab.vet.cornell.edu/resources/Chemical-Resistance-Chart-Detail.pdf