A+ 생화학실험 <5주차. Transformation> 레포트

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1. Competent cell (DH5α)

DH5α는 recA1 유전자의 160번 아미노산이 glycine에서 aspartic acid로 변이되는 돌연변이로 인하여 endonuclease의 기능을 상실하고 재조합 효소의 활성을 억제되었다. 이에 삽입된 plasmid DNA가 세포 내에서 안정적으로 유지되고 분해되지 않도록 하여, 높은 transformation efficiency(1×10^8 cfu/μg)를 가진다.
2. Plasmid DNA

Plasmid DNA는 박테리아의 chromosomal DNA와 물리적으로 분리되어 있으면서 자체 복제 능력을 가진 extrachromosomal DNA 분자이다. 인공 plasmid DNA는 DNA cloning 분야에서 vector로써 활용된다. Plasmid DNA에는 특정 DNA 조각을 삽입할 수 있는 site를 포함하고 있기에, 목표로 하는 DNA를 plasmid DNA에 삽입한 후 이를 박테리아의 자체 복제 과정을 통해 증폭, 복제, 발현시켜 목표 DNA를 다량 생산한다.
3. Ampicillin resistance

Ampicillin은 penicillin 계열에 속하는 antibiotics의 일종이다. Ampicillin은 박테리아가 분열할 때 필수적인 세포벽의 합성을 방해함으로써 살균 작용을 한다. Ampicillin에 대한 저항성을 가진 박테리아는 ampicillin이 존재하는 환경에서도 성공적으로 세포 분열을 수행하여 colony를 형성할 수 있다.
4. Transformation

Transformation은 세포 내로 외부 DNA가 들어가 유전 형질에 변화가 생기는 현상을 말한다. 이는 자연 상태에서 horizontal gene transfer의 주요 메커니즘 중 하나이며, 실험실 환경에서는 유전자 cloning 및 다양한 생명공학 연구에 필수적인 기술이다. 일반적으로 transformation의 효율을 높이기 위하여 competence cell을 사용한다.
5. Transformation efficiency

Transformation efficiency는 외부의 DNA가 competent에 성공적으로 도입되어 발현되는 정도를 나타내는 지표이다. 계산하는 방법은 Transformation efficiency = # CFU / DNA spread on the plate (μg)이다. Transformation efficiency가 높다는 것은 적은 양의 DNA로 많은 수의 cell이 성공적으로 변형되었음을 의미한다.
6. Serial dilution

Serial dilution은 특정 용액의 농도를 체계적으로 낮추기 위해 사용하는 방법으로, 각 단계에서 용액의 일정 부분을 취하여 새로운 용매에 추가함으로써 용액을 순차적으로 희석한다. 이를 통해 정확한 colony 수를 계수할 수 있다.
7. Plating

Plating은 cell이나 박테리아 샘플을 고체 medium 위에 배양하는 방법이다. Plating에는 streaking과 spreading 두 가지 주요 방법이 있다. Streaking은 주로 미생물의 정성분석에 쓰이는 방법이고, spreading은 colony 수를 정량적으로 계수할 때 사용한다.
8. SOC

SOC는 'Super Optimal broth with Catabolite repression'의 약자이며, SOB medium에 포도당을 추가한 것이다. SOC medium은 transformation 후 세포의 회복을 촉진하여 transformation efficiency를 높인다.
9. Transformation protocol

Transformation protocol에는 chemical transformation과 electroporation이 있다. Chemical transformation은 세포의 투과성을 증가시켜 세포 내로 외부 DNA를 운반하는 방식이고, electroporation은 cell에 고전압의 전기 충격을 가함으로써 세포막에 일시적인 구멍을 형성하여 DNA가 들어가게 하는 방식이다.
10. Transformation result analysis

본 실험에서 계산한 transformation efficiency 값은 A, B, C 각각 725 cfu/μg, 4535 cfu/μg, 9070 cfu/μg로, DH5α의 transformation efficiency 값인 1×10^8 cfu/μg보다 매우 낮은 값이었다. 이는 competent cell의 상태, DNA의 품질 및 양, 플레이팅 과정 등에서 문제가 있었을 가능성이 있다.

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1. Competent cell (DH5α)

Competent cells, such as DH5α, are a crucial component in molecular biology and genetic engineering. These cells have been genetically modified to be highly receptive to foreign DNA, making them ideal for transformation experiments. DH5α cells are commonly used due to their high transformation efficiency, which allows for the successful incorporation of plasmid DNA into the bacterial cells. The ability of these cells to readily take up and maintain plasmid DNA is essential for various applications, including gene cloning, protein expression, and genetic manipulation. Understanding the properties and handling of competent cells like DH5α is fundamental to many molecular biology techniques and experiments.
2. Plasmid DNA

Plasmid DNA is a circular, extrachromosomal genetic element that is widely used in molecular biology and genetic engineering. Plasmids are often employed as vectors to introduce foreign DNA into host cells, such as bacteria, for various purposes. They typically contain essential elements like origins of replication, selectable markers (e.g., antibiotic resistance genes), and multiple cloning sites that facilitate the insertion and manipulation of target genes. The ability to isolate, purify, and manipulate plasmid DNA is a core skill in molecular biology, enabling researchers to study gene expression, perform genetic modifications, and produce recombinant proteins. Understanding the properties, structure, and applications of plasmid DNA is crucial for many experimental procedures in the field of biotechnology and genetic research.
3. Ampicillin resistance

Ampicillin resistance is a commonly used selectable marker in molecular biology and genetic engineering. Many plasmids are engineered to confer ampicillin resistance to the host cells, allowing for the selection and identification of successfully transformed bacteria. This resistance is typically achieved by the inclusion of the β-lactamase gene, which encodes an enzyme that inactivates the ampicillin antibiotic. The presence of this resistance marker enables researchers to selectively grow and propagate only the transformed cells, as the untransformed cells will not survive in the presence of ampicillin. Understanding the mechanism and applications of ampicillin resistance is essential for the successful execution of various cloning, expression, and screening experiments involving plasmid DNA.
4. Transformation

Transformation is a fundamental technique in molecular biology and genetic engineering, where foreign DNA is introduced into a host cell, such as bacteria, to confer new genetic properties. This process involves the uptake of plasmid DNA by competent cells, which are cells that have been made permeable to allow the entry of exogenous DNA. Successful transformation leads to the integration of the plasmid DNA into the host cell, enabling the expression of the genes carried by the plasmid. Transformation is a crucial step in many applications, including gene cloning, protein expression, and genetic manipulation. Understanding the principles, protocols, and factors affecting transformation efficiency is essential for the successful execution of a wide range of molecular biology experiments.
5. Transformation efficiency

Transformation efficiency is a crucial parameter in molecular biology and genetic engineering, as it determines the success rate of introducing foreign DNA into host cells. Transformation efficiency is typically measured as the number of colony-forming units (CFUs) per microgram of plasmid DNA used in the transformation process. A high transformation efficiency is desirable, as it increases the likelihood of obtaining the desired transformants and facilitates the subsequent steps in genetic manipulation and analysis. Factors that can influence transformation efficiency include the competence of the host cells, the quality and concentration of the plasmid DNA, the transformation method used, and the optimization of the transformation protocol. Understanding and maximizing transformation efficiency is essential for the successful execution of various molecular biology techniques, such as gene cloning, protein expression, and genetic engineering.
6. Serial dilution

Serial dilution is a fundamental technique in molecular biology and microbiology, used to prepare a series of dilutions of a sample or solution. This technique is particularly important in the context of transformation experiments, where it is used to determine the transformation efficiency by counting the number of colony-forming units (CFUs) on agar plates. By performing a serial dilution of the transformed bacterial cells, researchers can obtain a range of dilutions that allow for accurate quantification of the transformed cells. This information is crucial for calculating the transformation efficiency and evaluating the success of the transformation process. Understanding the principles and proper execution of serial dilution is essential for various applications, including microbial enumeration, enzyme activity assays, and the preparation of standard curves in analytical techniques.
7. Plating

Plating is a crucial step in transformation experiments, where the transformed bacterial cells are spread onto agar plates containing a selective medium. This process allows for the isolation and enumeration of the successfully transformed cells, which are able to grow and form colonies on the plates. The selective medium, often containing an antibiotic like ampicillin, ensures that only the transformed cells, which carry the resistance gene, are able to survive and proliferate. Proper plating techniques, such as ensuring an even distribution of the cells and using the appropriate volume of the cell suspension, are essential for obtaining accurate counts of the transformed colonies and calculating the transformation efficiency. Understanding the principles and best practices of plating is a fundamental skill in molecular biology and microbiology, as it enables the successful isolation and analysis of genetically modified organisms.
8. SOC

SOC (Super Optimal broth with Catabolite repression) is a specialized growth medium used in transformation experiments, particularly for the recovery and outgrowth of transformed bacterial cells. After the introduction of plasmid DNA into competent cells, the SOC medium provides the necessary nutrients and conditions for the cells to recover, express the antibiotic resistance gene, and begin dividing. The SOC medium is typically used for a short incubation period (e.g., 1 hour) before the cells are plated on selective agar plates. The components of SOC, such as glucose and various salts, support the metabolic processes and growth of the transformed cells, enhancing their survival and the overall transformation efficiency. Understanding the role and composition of SOC medium is crucial for optimizing the transformation protocol and ensuring the successful recovery and propagation of the transformed bacterial cells.
9. Transformation protocol

The transformation protocol is a standardized set of steps and procedures used to introduce foreign DNA, such as plasmids, into competent host cells, typically bacteria. This protocol involves various stages, including the preparation of competent cells, the mixing of the cells with the plasmid DNA, the heat shock or other methods to facilitate DNA uptake, and the subsequent recovery and plating of the transformed cells. The transformation protocol must be carefully followed to ensure the highest possible transformation efficiency and the successful integration of the plasmid DNA into the host cells. Understanding the principles, best practices, and potential pitfalls of the transformation protocol is essential for researchers working in the fields of molecular biology, genetic engineering, and biotechnology, as it underpins many fundamental techniques and experiments.
10. Transformation result analysis

Analyzing the results of a transformation experiment is a crucial step in evaluating the success and efficiency of the process. This analysis typically involves quantifying the number of transformed colonies that grow on selective agar plates, as well as assessing the quality and characteristics of the transformed cells. Key parameters to analyze include the transformation efficiency, calculated as the number of colony-forming units (CFUs) per microgram of plasmid DNA, and the phenotypic or genotypic properties of the transformed cells, such as the expression of the desired recombinant protein or the presence of the correct plasmid DNA. Careful analysis of the transformation results allows researchers to optimize the protocol, troubleshoot any issues, and ensure the successful execution of downstream experiments involving the transformed cells. Understanding the principles and best practices of transformation result analysis is essential for researchers working in the fields of molecular biology, genetic engineering, and biotechnology.

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