基于SsrA/NIa的革兰阴性菌蛋白质水平翻译后调控策略

Gonzalo Durante-Rodríguez; Belén Calles; Víctor  de Lorenzo; Pablo I. Nikel

doi:10.21769/BioProtoc.3688

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Peer-reviewed

A SsrA/NIa-based Strategy for Post-Translational Regulation of Protein Levels in Gram-negative Bacteria

基于SsrA/NIa的革兰阴性菌蛋白质水平翻译后调控策略

Gonzalo Durante-Rodríguez email

BC Belén Calles

Víctor de Lorenzo

PN Pablo I. Nikel

发布: 2020年07月20日第10卷第14期 DOI: 10.21769/BioProtoc.3688 浏览次数: 3313

评审: Alba BlesaLi ZhangThibaud T. Renault

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Abstract

Strategies to control the levels of key enzymes of bacterial metabolism are commonly based on the manipulation of gene of interest within the target pathway. The development of new protocols towards the manipulation of biochemical processes is still a major challenge in the field of metabolic engineering. On this background, the FENIX (functional engineering of SsrA/NIa-based flux control) system allows for the post-translational regulation of protein levels, providing both independent control of the steady-state protein amounts and inducible accumulation of target proteins. This strategy enables an extra layer of control over metabolic fluxes in bacterial cell factories (see Graphical abstract below). The protocol detailed here describes the steps needed to design FENIX-tagged proteins and to adapt the system to virtually any pathway for fine-tuning of metabolic fluxes.

Graphical abstract

Keywords: Synthetic biology (合成生物学)

Metabolic engineering (代谢工程)

Proteolysis (蛋白质水解)

Escherichia coli (大肠杆菌)

Pathway engineering (途径工程)

Background

Controlling protein production has become a challenging problem that has been mostly tackled by manipulating the regulation of its production at different levels, such as the DNA level or the amount of protein(s) produced (Wu et al., 2016; Avcilar-Kucukgoze et al., 2017). At the DNA level, both transcription and translation have been widely studied in several microorganisms, enabling the design and development of a large number of synthetic circuits to improve bioproduction processes (Guzmán et al., 1995; Lutz and Bujard, 1997). In contrast, the protein level has been much less studied, with protocols mainly based on RNA interference, riboregulators and specific transcriptional regulators (Isaacs et al., 2004; Lou et al., 2012).

In order to implement a new approach towards the regulation of protein levels, we recently proposed a synthetic biology tool called FENIX (functional engineering of SsrA/NIa-based flux control) (Durante-Rodríguez et al., 2018). FENIX is based on the activity of the protease NIa, capable of recognizing and cleaving a specific sequence of 13 amino acids (the nia target, GESNVVVHQADER) (Verchot et al., 1991; Stevens, 2000; Calles et al., 2019), and the action of the proteasome, which recognizes a sequence of 11 amino acids located in the C-terminal region of a protein (the ssrA target, AANDENYALAA) (Doma and Parker, 2007; Shoemaker et al., 2010). We have designed a synthetic NIa/SsrA tag (GESNVVVHQADER•AANDENYALAA) that can be easily fused to the C-terminal module of any protein of interest via a single cloning step in a standardized vector. The FENIX system relies on the constitutive degradation of the target protein by the proteasome, followed by conditionally restoring protein accumulation by the cleavage of the proteasome tag (ssrA) through the action of the protease NIa. The whole circuit is triggered by the addition of 3-methylbenzoate, which induces the XylS/Pm expression system, thus controlling the individual components of the FENIX system. This novel tool can be also used in Pseudomonas putida for tight control of protein accumulation (Volke et al., 2020). The protocol described below discusses the overall strategy along with specific details on its implementation.

Materials and Reagents

Kits
1. High-Pure plasmid isolation kit (Roche, catalog number: 11754777001 )
2. Gene-Clean Turbo kit (Q-BIOgene, catalog number: 1102-400 )
Enzymes
1. Pfu DNA polymerase (Promega, catalog number: M7741 )
2. T4 DNA ligase (New England Biolabs, catalog number: M0202L )
3. Restriction enzyme NheI (New England Biolabs, catalog number: R3131S )
4. Restriction enzyme BsrGI (New England Biolabs, catalog number: R3575S )
5. Restriction enzyme SpeI (New England Biolabs, catalog number: R3133L )
Chemicals and other consumables
1. 0.22 μm filter membranes (Merck, catalog number: GSWP02500 )
2. Multiwell microtiter plates, 96-well, Flat Bottom (Falcon, catalog number: 353072 )
3. 50 ml plastic tubes (Falcon)
4. 2-ml microcentrifuge tubes
5. 1.5-ml microcentrifuge tubes
6. Aluminum foil
7. Petri dishes
8. Chloramphenicol (Sigma-Aldrich, catalog number: C0378-25G )
9. Kanamycin sulfate (Roche, catalog number: 10106801001 )
10. Absolute ethanol (Sigma-Aldrich, catalog number: 1009832500 )
11. 85% (v/v) glycerol (Sigma-Aldrich, catalog number: 1040942500 )
12. NaCl (Merck, catalog number: 1.06404.1000 )
13. NaOH (Merck, catalog number: 1.06498.0500 )
14. CaCl₂ (Merck, catalog number: 1.02382.1000 )
15. RbCl (Sigma-Aldrich, catalog number: R2252-100G )
16. MnCl₂ (Merck, catalog number: 1.05927.0100 )
17. Potassium acetate (Merck, catalog number: 1.04820.1000 )
18. Acetic acid (glacial) (Merck, catalog number: 1.00063.1000 )
19. MOPS (Sigma-Aldrich, catalog number: M1254-250G )
20. 3-methylbenzoate (Sigma-Aldrich, catalog number: 89890 )
21. Agar (Conda, catalog number: 1806 )
22. Agarose D1 (Conda, catalog number: 8019.22 )
23. Bacto^TM tryptone (BD, catalog number: 211705 )
24. Bacto^TM yeast (BD, catalog number: 212750 )
25. Competent E. coli DH10B cells (see Recipes)
26. Solution for DH10B competent cells TBFI (see Recipes)
27. Solution for DH10B competent cells TBFII (see Recipes)
28. 30 mg/ml chloramphenicol and 50 mg/ml kanamycin (see Recipes)
29. LB broth (see Recipes)
30. LB agar with 30 μg/ml chloramphenicol and/or 50 μg/ml kanamycin (see Recipes)
31. 0.5 M 3-methylbenzoate (see Recipes)
32. 50% (v/v) and 70% (v/v) ethanol (see Recipes)

Equipment

ABI Prism 377 automated DNA sequencer (Applied Biosystems Inc.)
Electroporator (Gene Pulser, Bio-Rad)
Orbital shaker
Flasks (50 ml, 1 L)
Pipettes (Gilson, models: PIPETMAN P2, P10, P20, P100, P200, P1000, P5000)
Centrifuge (Tomy, model: MX-301 )
Thermal Cycler (Applied Biosystems, model: 2720Thermal Cycler )
Temperature chamber (Taitec, model: Thermo minder SM-10R )
Water bath shaker (Taitec, model: Personal-11 )
Plate reader (Corona, model: MTP-880Lab )
Autoclave (Tomy Seiko, model: LSX-500 )

Software

Microsoft Excel (Microsoft)
BioEdit Sequence Alignment Editor
ApE-A plasmid Editor (https://jorgensen.biology.utah.edu/wayned/ape)

Procedure

English

中文翻译

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Durante-Rodríguez, G., Calles, B., Lorenzo, V. D. and Nikel, P. I. (2020). A SsrA/NIa-based Strategy for Post-Translational Regulation of Protein Levels in Gram-negative Bacteria. Bio-protocol 10(14): e3688. DOI: 10.21769/BioProtoc.3688.