Part:BBa_K4417007

CuO Cumate Inducible System

Description

This part is a novel cumate inducible promoter. This promoter was first synthesized by Claudia Schmidt-Dannert lab to induce the production of sfGFP.

Usage and Biology

Cumate, as an inducer, is non-toxic at working concentrations, less expensive than IPTG, and not dependent on a carbon source, making it a cost-effective and efficient method for inducing protein expression in large-scale industrial production. p-isopropyl benzoate (cumate) inducible gene expression system was constructed by combining the strong constitutive Bacillus promoter P_veg with regulatory elements of the Pseudomonas putida, CymR repressor, and CuO operator sequence. CymR was expressed by a weak P_xylR promoter, allowing the OFF and ON switch of an inducible sfGFP reporter under the cumate-inducible promoter. In the absence of cumate, the CymR repressor will bind to the CuO operator and block sfGFP production from the P_veg promoter. With cumate, the CymR will be released from the CuO operator; therefore, the sfGFP expression will be switched on.

Figure 1: Inducible cumate promoter expression system.

Research literature indicates three important features of this promoter:

• Higher protein expression levels in Bacillus compared to other reported inducible Bacillus expression systems.
• Also inducible in E. coli.
• Tuneable protein expression levels by varying cumate concentration.

Method

Cumate is non-toxic to the host. In our experiment, we tried to induce the sfGFP expression in E. coli with different cumate concentrations (10-100 μM).

Characterization

Cumate testing

Cumate promoter function was evaluated by cumate testing. 5 mL cultures of DH5-α transformed with pCT5c were induced with 50 µM and 100 µM cumate, and an un-induced control sample was included. Overexpression of sfGFP was visible by eye when collecting the cell pellets following 15hrs incubation with cumate at 37°C, as shown in Fig 2.

Figure 2: Cell pellets overexpressing sfGFP with cumate induction after 24hrs. The Colour change of the pellet from light brown to green was observed. Uninduced control cultures (-) do not express sfGFP, and no change in colour was seen.

Fluorescence measurements of sfGFP are shown in Figure 3. From a single culture, cells were incubated overnights and split into 5 parts, four of them were induced with different cumate concentrations (10 µM, 30 µM, 50 µM, and 70 µM) and one was not induced. Induced DH5-α pCT5 cultures were strongly fluorescent compared to the non-induced DH5-α pCT5 (~10-fold with 50 µM cumate and ~6-fold with 10 µM cumate).

Figure 3: Cumate testing in DH5-α transformed with pCT5c. Fluorescence reading from 0-10 hrs.

The growth rate of WT E. coli was measured at OD₉₀₀ as shown in Figure 4.

Figure 4: Growth curve of transformed E. coli in cumate testing.

Reference

1. Development of a synthetic cumate-inducible gene expression system for Bacillus. Seo SO, Schmidt-Dannert C. Appl Microbiol Biotechnol. 2018 Nov 3. pii: 10.1007/s00253-018-9485-4. doi: 10.1007/s00253-018-9485-4. 10.1007/s00253-018-9485-4 PubMed 30392122

2. Addgene plasmid # 119872; http://n2t.net/addgene:119872; RRID:Addgene_119872

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 1
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]