蓝藻菌鱼腥藻属物种 PCC 7120中的靶标基因失活

Kangming Chen; Huilan Zhu; Liping Gu; Shengni Tian; Ruanbao Zhou

doi:10.21769/BioProtoc.1890

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Target Gene Inactivation in Cyanobacterium Anabaena sp. PCC 7120

蓝藻菌鱼腥藻属物种 PCC 7120中的靶标基因失活

Ruanbao Zhou email

发布: 2016年08月05日第6卷第15期 DOI: 10.21769/BioProtoc.1890 浏览次数: 10290

评审: Maria SinetovaClaudia CatalanottiYoko Eguchi

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Abstract

Anabaena sp. strain PCC 7120 has long served as a model organism for investigating N₂-fixation, photosynthesis, and various plant-type metabolic pathways and biofuel production, as well as cellular differentiation (Xu et al., 2008, Halfmann et al., 2014, Golden and Yoon, 2003). Since more than 30,000 sequenced bacterial genomes are currently available (Land et al., 2015), specific gene inactivation and analyses of the corresponding mutant’s phenotype have become powerful tools in elucidating the function of a target gene. Here we describe a protocol to inactivate a target gene in Anabaena sp. PCC 7120 using a single-crossover approach. This approach requires only one-step cloning of an internal fragment of a target gene into an integrative vector to produce a cargo plasmid. Upon a single crossover (homologous recombination) between the cargo plasmid and the Anabaena chromosome, the endogenous target gene is disrupted by generating 3’- and 5’-deleted fragments. This gene inactivating protocol is based on an integrative vector pZR606 (Chen et al., 2015), which may be broadly applied to gene inactivation in other cyanobacterial species as well as other prokaryotic organisms.

Keywords: Genetic tools for bacteria (细菌的遗传工具)

Genetic engineering (遗传工程)

Integration vector method (整合载体的方法)

Single cross-over method (单交叉法)

Synthetic cyanobacteria (合成的蓝藻)

Materials and Reagents

Petri dishes (Fisher Scientific, catalog number: FB0875713 )
Glass flask (Fisher Scientific, catalog number: FB-500-50 )
1.5 ml centrifuge tubes (Fisher Scientific, catalog number: 02-682-550 )
15 ml conical centrifuge tubes (Fisher Scientific, catalog number: 05-527-90 )
50 ml conical centrifuge tubes (Fisher Scientific, catalog number: 12-565-270 )
Anabaena sp. strain PCC 7120 (hereafter Anabaena 7120)
Escherichia coli (E. coli) NEB10β [Δ(ara-leu) 7697 araD139 fhuAΔlacX74 galK16 galE15 e14-Φ80dlacZΔM15 recA1 relA1 endA1 nupG rpsL(StrR)rph spoT1Δ(mrr-hsdRMS-mcrBC); New England BioLabs, catalog number: C3019H ]
E. coli HB101 [F^-mcrB mrr hsdS20(r_B^-m_B^-) recA13, leuB6, ara-14, proA2, lacY1, galK2, xyl-5, mtl-1, rpsL20(Sm^R) glnV44 λ^- ; Promega, catalog number: L2015 ]
Conjugal plasmid pRL443 and helper plasmid pRL623 (Elhai et al., 1997)
pZR606, an integrative vector for Anabaena 7120 (Chen et al., 2015). pZR606 is available upon request (GenBank, catalog number: KJ500179.1 )
pZR670 (available upon request), a replicative vector in Anabaena 7120 (Xu et al., 205) [The map of pZR670 is provided (Figure S1).]
LB broth (Sigma-Aldrich, catalog number: L3522-1KG )
LB agar, used for growing E. coli (MP, catalog number: 100262 )
Note: This brand of agar does not work for cyanobacteria.
Agar, required for growing Anabaena 7120 and other cyanobacteria (Fisher Scientific, catalog number: A360-500 )
Immobilon-NC transfer membrane (Millipore, catalog number: HATF08550 )
Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518-25G )
Chloramphenicol (Fisher Scientific, catalog number: BP904-100 )
Erythromycin (Sigma-Aldrich, catalog number: E6376-25G )
Kanamycin sulfate (Sigma-Aldrich, catalog number: K4000-25G )
Spectinomycin dihydrochloride pentahydrate (Sigma-Aldrich, catalog number: S9007-25G )
MgSO₄·7H₂O (MP, catalog number: 194833 )
CaCl₂·2H₂O (Fisher Scientific, catalog number: BP510-500 )
NaCl (Fisher Scientific, catalog number: S271-1 )
K₂HPO₄ (Fisher Scientific, catalog number: BP363-500 )
KNO₃ (Fisher Scientific, catalog number: BP368-500 )
NaNO₃ (Fisher Scientific, catalog number: BP360-500 )
MnCl₂·4H₂O (Fisher Scientific, catalog number: M87-100 )
Na₂Mo₄·2H₂O (99% purity) (Acros Organics, catalog number: 206371000 )
ZnSO₄·7H₂O (Fisher Scientific, catalog number: Z76-500 )
CuSO₄·5H₂O (Fisher Scientific, catalog number: BP346-500 )
H₃BO₃ (Fisher Scientific, catalog number: BP168-500 )
NH₄VO₃ (Acros Organics, catalog number: 194910500 )
CoCl₂·6H₂O (Fisher Scientific, catalog number: C371-100 )
KOH (Fisher Scientific, catalog number: P250-500 )
Na₂EDTA·2H₂O (Fisher Scientific, catalog number: BP120-500 )
FeSO₄·7H₂O (Fisher Scientific, catalog number: I146-500 )
Allen and Arnon medium plus nitrate: AA/8(N) (see Recipes)

Equipment

Centrifuges (Beckman, model: Allegra X-15R ; Thermo Fisher Scientific, Sorvall^TM Legend^TM, model: Micro17 )
Innova-44R shaker equipped with continuous fluorescent light illumination (ca. 50-100 μE/m²s) (Eppendorf, New Brunswick^TM Innova^®, model: 44R )
Cyanobacteria Culture Room (constant at 30 °C) equipped with a digital temperature controlling system and Sylvania fluorescent light bulbs (F40CWX, 40W/4100K, T12)
Note: Light shelves in cyanobacteria culture room are able to provide continuous light illumination (ca. 50-150 μE/m²s).
ESCO laminar flow cabinet (ESCO, Clean BenchAirstream^® model: AHC-4B2 )
Water bath sonicator (Bransonic^® Ultrasonic Cleaner, model: 1510R-MT )
Microscope, Olympus upright compound microscope (Olympus, model: AX70; BX53 )
PCR thermal cycler (Bio-Rad Laboratories, Touch^TM, model: C1000 )
UV-Vis spectrophotometer (Thermo Fisher Scientific, model: GeneSyS 10S )

Procedure

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Chen, K., Zhu, H., Gu, L., Tian, S. and Zhou, R. (2016). Target Gene Inactivation in Cyanobacterium Anabaena sp. PCC 7120. Bio-protocol 6(15): e1890. DOI: 10.21769/BioProtoc.1890.