通过共轭技术将质粒从细菌转移到硅藻中

Federico  Berdun; Matías  Valiñas; Gabriela Pagnussat; Eduardo  Zabaleta

doi:10.21769/BioProtoc.4945

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Peer-reviewed

Mobilization of Plasmids from Bacteria into Diatoms by Conjugation Technique

通过共轭技术将质粒从细菌转移到硅藻中

FB Federico Berdun

MV Matías Valiñas

Gabriela Pagnussat

EZ Eduardo Zabaleta email

发布: 2024年03月05日第14卷第5期 DOI: 10.21769/BioProtoc.4945 浏览次数: 2115

评审: Noelia ForesiEmilia KrypotouIsmail Tahmaz

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The authors used this protocol in:

Cover of Molecular Microbiology, featuring study using the protocol.

Feb 2021

Special Issue on Protocols for Organoid Research

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

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Abstract

Diatoms serve as a source for a variety of compounds with particularbiotechnological interest. Therefore, redirecting the flow to a specific pathwayrequires the elucidation of the gene’s specific function. The mostcommonly used method in diatoms is biolistic transformation, which is a veryexpensive and time-consuming method. The use of episomes that are maintained asclosed circles at a copy number equivalent to native chromosomes has become auseful genetic system for protein expression that avoids multiple insertions,position-specific effects on expression, and potential knockout of non-targetedgenes. These episomes can be introduced from bacteria into diatoms viaconjugation. Here, we describe a detailed protocol for gene expression thatincludes 1) the gateway cloning strategy and 2) the conjugation protocol for themobilization of plasmids from bacteria to diatoms.

Keywords: Conjugation (共轭)

Diatom (硅藻)

Bacteria (细菌)

Mobilization plasmid (质粒迁移作用)

Destination vector (目的载体)

Phaeodactylum tricornutum (三角褐指藻)

Background

Diatoms are unicellular, predominantly photosynthetic, free-living microorganisms that play a central role in trophic webs as primary producers [1]. Diatoms represent the most abundant group within the phytoplankton community, contributing to 40% of global CO₂ fixation in the oceans. These organisms are found in freshwater bodies, making them one of the most ecologically successful microalgae worldwide [2,3]. One physiological aspect that explains its ecological success is the energetic-metabolic coupling between mitochondria and chloroplasts [4,5].

From a biotechnological point of view, these organisms produce a variety of compounds of interest such as silica frustule, used as filtering and abrasive materials, and terpenes like fucoxanthin, lupeol, and betulin with antitumoral and antioxidant properties [6]. Furthermore, these organisms are a source of traditional biofuels including methane, through the anaerobic digestion of algae biomass, and biodiesel derived from oil. These organisms show an interesting lipid profile, rich in very long– chain polyunsaturated fatty acids (ω-3 and ω-6), which are of great interest to the industry, especially because they are not usual in other organisms like chlorophytes and flowering plants [7].

The advancement of genetic tools and increasing knowledge on metabolic pathways have facilitated the development of strategies to enhance the productivity of diatoms byredirecting the metabolic flow towards desired products [8]. Thus, diatom genetic manipulation is essential for the elucidation of specific gene functions. Biolistic transformation methods are standard for many diatom species; however, they are time-consuming and require high-yield plasmid DNA preparations and access to expensive specific equipment and reagents (e.g., gene gun). On the other hand, episodes provide a reliable, consistent, and predictable platform for protein expression by avoiding the complications of random chromosomal integration including multiple insertions, position-specific effects on expression, and potential knockout of non-targeted genes. They can be efficiently transferred from bacteria into the diatom via the conjugation method developed by Karas et al. [9] and improved by Diner et al. [10]. Here, we thoroughly describe the procedure previously published [11] and frequently employed in our lab for cloning genes of interest and the subsequent mobilization of vector constructions from bacteria into diatoms by conjugation.

Materials and reagents

Biological materials

Pipette tips: 1–2 µL, 2–200 μL, and 100–1,000 μL (Deltalab, catalog number: 200070 and 301-09)
90 mm diameter Petri dishes (Deltalab, catalog number: 200209)
1.5 mL microcentrifuge tubes (Deltalab, catalog number: 200400P)
50 mL conical centrifuge tubes (Deltalab, catalog number: 42993)
0.22 μm nylon membrane filters (e.g., GVS, catalog number: FJ13BNPNY002AD01)

Reagents

Phaeodactylum tricornutum liquid cultures [e.g., Culture collection of algae, University of Texas, Austin (Utex), catalog number: 646]
PCR kit
1. dNTPs (Promega, catalog number: U123A)
2. Platinum Pfx (Invitrogen, catalog number: 11708-013)
3. 10× Pfx amplification buffer (Invitrogen, catalog number: 52806)
4. MgCl₂ 25 mM (Promega, catalog number: A351H)
5. Taq Pegasus (Productos Bio-Logicos, catalog number: EA01M)
6. GoTaq green buffer 5× (Promega, catalog number: M791A)
Destination plasmid pFcpB (Addgene, catalog number: 90098)
pTA-Mob (Addgene, catalog number: 149662)
Escherichia coli DH10B (Thermo Fisher, catalog number: Eco 113)
E. coli pTA-Mob liquid cultures (homemade)
pENTR/D-TOPO Vector kits (Thermo Fisher Scientific, catalog number: K240020SP)
LR Clonase Enzyme mix (Thermo Fisher Scientific, catalog number: 11791019)
Agar medium 2% (Britannia, catalog number: B0101406)
Luria Broth (LB) medium (homemade)
Gentamicin (Gn) stock solution 25 mg/mL (1:1,000) (Merck, catalog number: G3632)
Kanamycin (Kn) stock solution 50 mg/mL (1:1,000) (Merck, catalog number: BP861)
Ampicillin (Amp) stock solution 100 mg/mL (1:1,000) (Merck, catalog number: A9518)
Zeocin (Zeo) stock commercial solution (Invitrogen, catalog number: R25001); use a concentration of 7.5 µL commercial solution stock in 10 mL of BG11 medium
Amphotericin (Amph) stock solution B 2.5 mg/ mL (1:1,000) (Richet S.A laboratory, https://www.richet.com.ar/en/list?t=n&i=13); use a concentration of 2.5 µg/ mL
BG11 medium (homemade)
Selection plate (0.5× BG11, 1% agar + antibiotics (Amp + Kn + Amph + Gn + Zeo)
Conjugation plate (0.5× BG11 + 0.9% agar + 5% LB + antibiotics (Amp + Amph + Gn)
Tryptone (BD, catalog number: 211705)
Yeast extract (Oxoid, catalog number: LP0021)
NaCl (J.T. Baker, catalog number: 3624-19)
Na₂Mg EDTA (Sigma-Aldrich, catalog number: 14402-88-1)
Ferric citrate (Merck, catalog number: 3522-50-7)
CaCl₂·2H₂O (Merck, catalog number: 10035-04-8)
MgSO₄·7H₂O (Cicarelli, catalog number: 1054214)
K₂HPO₄ (Cicarelli, catalog number: 1015214)
H₃BO₃ (Cicarelli, catalog number: 771214)
MnCl₂·4H₂O (Merck, catalog number: 13446-34-9)
ZnSO₄·7H₂O (Merck, catalog number: 7446-20-0)
CuSO₄·5H₂O (Merck, catalog number: 1.02790.1000.1026)
CoCl₂·6H₂O (Merck, catalog number, 7791-13-1)
Na₂MoO₄·2H₂O (Merck, catalog number: 10102-40-6)
NaCO₃ (Merck, catalog number: 497-19-8)
NaNO₃ (Merck, catalog number: 7631-99-4)

Solutions

LB medium (1 L)
BG11 medium (1 L):
Stock I (1 L)
Stock II (1 L)
Stock III (1 L)
Stock V - microelements (1 L)
Carbonate supplement (50 mL)
Nitrate supplement (50 mL)

Recipes

LB medium (1 L)
10 g of tryptone
5 g of yeast extract
10 g of NaCl
1 L of dH₂O
BG11 medium (1 L)
Stock I (1 L):
0.1 g of Na₂Mg EDTA
0.553 g of ferric citrate
3.6 g of CaCl₂·2H₂O
Filter sterilize into a sterile bottle or autoclave.

Stock II (1 L):
7.5 g of MgSO₄·7H₂O
Filter sterilize into a sterile bottle or autoclave.

Stock III (1 L):
3.05 g of K₂HPO₄
Filter sterilize into a sterile bottle or autoclave.

Stock V - microelements (1 L):
2.86 g of H₃BO₃
1.81 g of MnCl₂·4H₂O
0.222 g of ZnSO₄·7H₂O
0.074 g of CuSO₄·5H₂O
0.05 g of CoCl₂·6H₂O
0.4451 g of Na₂MoO₄·2H₂O
Filter sterilize into a sterile bottle or autoclave.

Carbonate supplement (50 mL)
1 g of NaCO₃
Filter sterilize into a sterile bottle.

Nitrate supplement (50 mL)
15 g of NaNO₃
Filter sterilize into a sterile bottle.

For basic BG11 (1 L), combine the following stock solutions:
Add 10 mL of stock I, II, and III.
Add 1 mL of stock V and carbonate supplement.
Add 5 mL of nitrate supplement.
Add sterilized dH₂O to complete to 1 L.

Equipment

Pipettes
Neubauer counting chamber
Laminar air flow equipment
Centrifuge with 50 mL tube capacity
Erlenmeyer
Room or chamber at 37 ºC
Room or chamber at 18 ºC with light 100-200 PAR
Shaker (Vicking, model: Shaker Pro)
Spectrophotometer (Gene Quant, model: 1300)
Binocular light microscope
Applied Biosystems Veriti Thermocycler

Procedure

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Berdun, F., Valiñas, M., Pagnussat, G. C. and Zabaleta, E. J. (2024). Mobilization of Plasmids from Bacteria into Diatoms by Conjugation Technique. Bio-protocol 14(5): e4945. DOI: 10.21769/BioProtoc.4945.