11주차_sequencing BL21 transformation

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1. Sanger sequencing

Sanger sequencing은 Frederick Sanger에 의해 1977년 개발된 방법으로, 500bp 이하의 DNA 서열을 결정할 때 99.99%의 매우 높은 정확도를 보인다. 이 방법은 DNA polymerase에 의한 DNA 중합 과정 중 template DNA 서열과 상보적인 염기가 결합된다는 성질을 이용하기 때문에 chain termination method 또는 dideoxynucleotide termination method라고 부르기도 한다. Sanger sequencing은 ddNTP를 이용하여 DNA polymerization을 중단시키고, 이렇게 중단된 DNA 분자들을 전기영동으로 분리하여 서열을 분석한다.
2. BL21(DE3) competent cell

BL21(DE3)는 λ prophage의 돌연변이인 DE3의 lysogen으로, 효율적인 단백질 발현을 위해 사용하는 competent cell이다. BL21(DE3)에는 T7 promoter가 존재하는 pET-16b에 필요한 T7 RNA polymerase가 존재하며, T7 RNA polymerase는 lacUV5 promoter의 조절을 받는다. 또한 Lon과 OmpT와 같은 protease가 존재하지 않기 때문에 단백질 발현에 매우 유리하다.
3. Transformation

이번 실험에서는 sequencing 결과를 바탕으로 emGFP가 성공적으로 들어간 gene을 BL21(DE3) competent cell에 transformation했다. Heat-shock을 이용하는 chemical transformation을 진행했으며, 1시간의 incubation 후 LB-agar plate에 spreading하여 37도에서 배양했다. 그 결과 22개의 큰 colony와 작은 colony들이 관찰되었고, 각각의 colony는 emGFP로 인해 녹색 빛을 나타냈다.

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1. Sanger sequencing

Sanger sequencing, also known as the chain-termination method, is a widely used DNA sequencing technique developed by Frederick Sanger in the 1970s. This method has played a crucial role in the advancement of molecular biology and genomics research. Sanger sequencing involves the synthesis of DNA strands complementary to the target DNA sequence, with the incorporation of fluorescently labeled dideoxynucleotides (ddNTPs) that terminate the DNA synthesis at specific positions. The resulting DNA fragments are then separated by size using gel electrophoresis, and the fluorescent signals are detected to determine the sequence of the target DNA. While newer sequencing technologies, such as next-generation sequencing (NGS), have surpassed Sanger sequencing in terms of throughput and cost-effectiveness, the Sanger method remains an important tool for various applications, including targeted gene sequencing, validation of NGS results, and sequencing of short DNA fragments. Its reliability, accuracy, and well-established protocols make it a valuable technique in many areas of molecular biology and genetics.
2. BL21(DE3) competent cell

BL21(DE3) competent cells are a widely used bacterial strain in molecular biology and protein expression experiments. These cells are derived from the Escherichia coli B strain and are engineered to express the T7 RNA polymerase, which is a key component in the T7 expression system. The T7 expression system is a powerful tool for the overexpression of recombinant proteins, as it utilizes the highly active T7 promoter to drive the transcription of the target gene. BL21(DE3) cells are particularly useful for the expression of proteins that may be toxic or difficult to express in other bacterial strains. The cells are designed to be competent, meaning they can readily take up and incorporate foreign DNA, such as plasmids containing the target gene. This makes them an efficient and convenient choice for protein expression experiments, as the transformation process is relatively straightforward. Overall, BL21(DE3) competent cells have become a staple in many molecular biology and protein engineering laboratories due to their versatility, efficiency, and ability to produce high levels of recombinant proteins.
3. Transformation

Transformation is a fundamental process in molecular biology and genetic engineering, where foreign DNA is introduced into a host cell, such as bacteria or eukaryotic cells. This process allows the host cell to express the genetic information encoded in the introduced DNA, enabling the production of desired proteins or the study of gene function. Transformation is a crucial step in many experimental procedures, including cloning, gene expression, and genetic manipulation. The process typically involves several steps, such as preparing competent cells, mixing the cells with the DNA of interest, and applying a method to facilitate the uptake of the DNA, such as heat shock or electroporation. Successful transformation results in the host cell incorporating the foreign DNA and expressing the encoded genes, which can then be selected and amplified for further experimentation. Transformation is a powerful tool that has enabled numerous advancements in fields like biotechnology, medicine, and basic research, as it allows researchers to manipulate and study genetic information in a controlled and efficient manner.

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