Optimization of Electroporation Conditions in Construction of Phage Display Antibody Library

doi:10.13523/j.cb.1910008

China Biotechnology

2020, Vol. 40

Issue (4): 42-48 DOI: 10.13523/j.cb.1910008

Optimization of Electroporation Conditions in Construction of Phage Display Antibody Library

YANG Li¹,SHI Xiao-yu²,LI Wen-lei²,LI Jian^2,^*(

),XU Han-mei^1,^*(

)

1 China Pharmaceutical University, Nanjing 211100, China
2 Tasly Biopharmaceuticals Co., Ltd., Shanghai 201203, China

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Abstract

Objective: To determine a stable and efficient electroporation protocol to construct a high-capacity phage display antibody library. Methods: Explored the effects of voltage, pulse time, quality and concentration of phagemid DNA, growth phase of TG1 E.coli, buffer for resuspension and washing and medium optimization on the electrotransformation efficiency of TG1 E.coli. Results: Using electroporation cuvettes with electrode spacing of 2 mm, the parameters of the electrorotator were set to 3kV, 25μF, 5ms, and 200 Ω. The exogenous DNA was purified and added to the competent bacterial suspension to make the final concentration of DNA 1ng/μl. 20mmol/L MgCl₂ was added to the medium, and the growth phase of TG1 was adjusted to OD₆₀₀=0.8. The cells were resuspended and washed with sterile ultrapure water, and the concentration of competent cells was adjusted to 4×10¹⁰ cells/ml. Under such conditions, the electrotransformation efficiency can reach 4.9×10⁹ CFU/μg DNA. Conclusion: Electrotransformation efficiency has been improved through multiple condition optimization, which established the basis for constructing a high-capacity phage display antibody library.

Key words： Electroporation Electrotransformation efficiency E.coli strain TG1 Phage display antibody library

Received: 10 October 2019 Published: 18 May 2020

ZTFLH:

Q936

Corresponding Authors: Jian LI,Han-mei XU E-mail: lijian16@tasly.com;1037714870@qq.com

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	Li YANG
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Cite this article:

YANG Li,SHI Xiao-yu,LI Wen-lei,LI Jian,XU Han-mei. Optimization of Electroporation Conditions in Construction of Phage Display Antibody Library. China Biotechnology, 2020, 40(4): 42-48.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.1910008 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I4/42

Fig.1 The electrophoresis results of TSL and pCANTAB 5E enzyme-digested product

Fig.2 The effects of voltage and pulse time on transformation efficiency

DNA 纯化方式	DNA 的量 (ng)	复苏菌液体积 (ml)	菌落数 (×10⁴ CFU, $x ?$ ±s)
未纯化	400	4.5	38.67±6.43
纯化	400	4.5	490.33±134.97

Table1 The effect of DNA purification on the quantity of transformants

Fig.3 The effect of DNA purification on transformation efficiency

DNA的浓度 (ng/μl)	DNA的量 (ng)	复苏菌液体积(ml)	菌落数 (×10⁵ CFU, $x ?$ ±s)
1	400	4.55	183.67±14.22
5	2 000	4.75	415.67±29.02
10	4 000	4.75	442.33±131.08

Table 2 The effect of DNA concentration on the quantity of transformants

Fig.4 The effect of DNA concentration on transformation efficiency

Fig.5 The effect of TG1 growth phase on transformation efficiency

感受态细胞制备条件	DNA的量 (ng)	复苏菌液体积 (ml)	菌落数 (×10⁵ CFU, $x ?$ ±s)
OD=0.5,甘油/甘露醇	100	1	36.00±7.21
OD=0.5,超纯水	100	1	291.33±34.43
OD=0.8,超纯水	100	1	368.00±63.93

Table 3 The effect of resuspension and washing buffer on the quantity of transformants

Fig.6 The effect of resuspension and washing buffer on transformation efficiency

培养基成分	DNA的量 (ng)	复苏菌液体积 (ml)	菌落数 (×10⁵ CFU, $x ?$ ±s)
无MgCl₂	100	1	240.00±60.70
含20 mmol/L MgCl₂	100	1	489.67±133.72

Table 4 The effect of MgCl₂ added in medium on the quantity of transformants

Fig.7 The effect of MgCl₂ added in medium on transformation efficiency


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