[생물공정실험] 6주차 P1 Transduction 결과보고서

문서 내 토픽

1. P1 Transduction

이번 실험에서는 P1 phage를 이용하여 recipient cell에 transduction하고 target 유전자가 삽입됐는지 colony 색으로 확인해보았다. E.Coli균의 사전 준비 과정은 조교님께서 대신 해주셨다. 이번 실험에서 recipient cell은 gadA gene이 knockout된 E.coliΔgadA이고, donor cell은 copA gene이 knockout되고 kanamycin 항생제에 대한 저항성을 가진 유전자가 삽입된 E.coliΔcopA::kan이다. 이를 통해 항생제에 대한 저항성을 가진 유전자가 제대로 삽입됐는지 확인하는 test를 진행했다.
2. P1 Phage

P1 phage는 DNA 나 RNA 유전물질을 보관하는 head랑 host virus에 부착하는 데 도움을 주는 tail로 구성된다. Phage는 오직 바이러스만 target하는데 P1 phage는 E.coli를 target한다. P1 trasnduction을 진행했는데 이는 박테리오파지 P1을 통해 host에서 다른 host로 유전정보를 전달하는 형질도입으로 유전자의 knockout을 생성해 그 유전자의 기능을 알기 위해 쓰였다.
3. Lysogenic Cycle vs Lytic Cycle

Phage life cycle은 lysogenic cycle과 lytic cycle로 나눠지는데 큰 차이점은 lysogenic은 박테리아가 죽지않고 공존하지만 lytic은 박테리아 안에서 증식한 후 host cell을 죽인다는 것이다. 즉 host virus에 adhesion하고 phage head의 유전정보를 주입하면 phage의 DNA와 host DNA가 homologous하게 recombination되는데 이를 가지고 살아가면 lysogenic, 이를 작은 단편으로 잘라 circular DNA로 만들어져 안에서 증식해 나가는 것을 lytic cycle이라 한다.
4. Calcium Ion and Cell Membrane

P1 salt에 칼슘이온이 포함된 이유는 E.coli의 세포막과 DNA가 음전하를 띠어 반발력이 생기므로 칼슘이온을 첨가해줌으로써 세포막 음전하를 중화시키고 일시적으로 막투과성을 변화시키는 방식으로 comepetent cell을 만들어주기 위함이다. 따라서 칼슘이온은 phage DNA가 cell안에 더 잘 들어가게 도와주는 것으로 사료된다.
5. EGTA

LB EGTA를 첨가했는데 이때 LB EGTA는 chelating agent로 칼슘을 co-factor로 사용하는 enzyme들을 block하기 위해 쓰인다. 즉 세포벽이 완전하게 유지되는 데 필수적인 칼슘이온을 제거해 세포벽이 군데군데 무너지게 한다. 따라서 LB EGTA를 사용해 phage가 E.coli가 다시 만나 중복감염을 일으키지 않게 한다.
6. EBU Plate

마지막으로 EBU plate에 spreading과정을 진행했는데 이는 selection하여 원하는 phenotype이 만들어졌는지 확인하기 위해 진행된다. EBU plate는 배지의 한 종류로 Evans blue와 Uranine이 포함되어있다. 만약 lytic cycle로 배양되면 E.coli를 이용해 파지가 갖는 단백질을 만들어서 숙주의 DNA를 절편으로 잘라지고 파지가 갖고 있는 것만 복제하며 E.coli cell을 파괴한다. 이때 박테리오파지에 의해 터져 나오는 내용물이 약산성을 띠므로 Evans blue 시약에 의해 colony가 파란색을 띠게 된다.

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1. P1 Transduction

P1 transduction is a process in which genetic material is transferred from one bacterial cell to another through the use of a bacteriophage, specifically the P1 phage. This process is an important mechanism for the spread of genetic information and the acquisition of new traits in bacteria. During P1 transduction, the P1 phage infects a bacterial cell, and instead of immediately lysing the cell and replicating, the phage genome integrates into the host's chromosome. When the host cell divides, the phage genome can be packaged into the new bacterial cells, effectively transferring genetic material from one cell to another. This process can lead to the acquisition of new genes, such as those conferring antibiotic resistance or virulence factors, which can have significant implications for the evolution and spread of bacterial pathogens. Understanding the mechanisms of P1 transduction is crucial for studying bacterial genetics, evolution, and the development of effective antimicrobial strategies.
2. P1 Phage

The P1 phage is a temperate bacteriophage that infects a wide range of Gram-negative bacteria, including Escherichia coli and Salmonella species. It is a well-studied model system for understanding the mechanisms of phage-host interactions and the genetic transfer processes mediated by phages. The P1 phage has a unique feature in that it can either undergo a lytic cycle, where it rapidly replicates and lyses the host cell, or a lysogenic cycle, where the phage genome integrates into the host's chromosome and is replicated along with the host's DNA. This ability to switch between these two life cycles allows the P1 phage to persist in the bacterial population and effectively spread its genetic material. Understanding the biology and genetics of the P1 phage has contributed to our broader knowledge of bacteriophages and their role in bacterial evolution, as well as their potential applications in areas such as phage therapy and genetic engineering.
3. Lysogenic Cycle vs Lytic Cycle

The lysogenic cycle and the lytic cycle are two distinct modes of reproduction employed by temperate bacteriophages, such as the P1 phage. In the lysogenic cycle, the phage genome integrates into the host's chromosome and remains dormant, replicating along with the host's DNA. This allows the phage to persist in the bacterial population without immediately causing cell lysis. In contrast, the lytic cycle involves the rapid replication of the phage genome, the assembly of new phage particles, and the eventual lysis of the host cell to release the newly formed phages. The choice between the lysogenic and lytic cycles is often influenced by environmental factors and the physiological state of the host cell. Understanding the differences between these two cycles is crucial for understanding the complex interactions between phages and their bacterial hosts, as well as the implications for bacterial evolution, pathogenesis, and potential phage-based therapeutic applications.
4. Calcium Ion and Cell Membrane

Calcium ions (Ca2+) play a crucial role in the structure and function of cell membranes. Calcium ions interact with the negatively charged phospholipids and proteins within the cell membrane, helping to maintain the membrane's structural integrity and stability. This interaction is particularly important in the regulation of membrane permeability, as calcium ions can influence the opening and closing of ion channels and the activity of membrane-bound enzymes. Additionally, calcium signaling pathways are involved in various cellular processes, such as cell signaling, neurotransmitter release, and muscle contraction. Disruptions in the regulation of calcium homeostasis can lead to various pathological conditions, highlighting the importance of understanding the role of calcium ions in cell membrane dynamics and cellular function. Studying the interplay between calcium ions and cell membranes is crucial for advancing our understanding of fundamental cellular processes and developing targeted therapeutic interventions for membrane-related disorders.
5. EGTA

EGTA (Ethylene Glycol-bis(β-Aminoethyl Ether)-N,N,N',N'-Tetraacetic Acid) is a chelating agent that is widely used in biological research and applications. Its primary function is to bind and sequester calcium ions (Ca2+) from the surrounding environment. EGTA has a high affinity for calcium ions, effectively removing them from solution and preventing them from participating in various cellular processes. This property makes EGTA a valuable tool in the study of calcium-dependent biological systems, as it allows researchers to manipulate the availability of calcium ions and investigate their role in cellular signaling, enzyme activity, and membrane dynamics. EGTA is commonly used in cell culture media, biochemical assays, and experimental protocols to control the calcium concentration and study the effects of calcium depletion on cellular function. Understanding the properties and applications of EGTA is crucial for researchers working in fields such as cell biology, neuroscience, and biochemistry, where the regulation of calcium homeostasis is of paramount importance.
6. EBU Plate

The EBU (Eosin-Methylene Blue) plate, also known as the EMB agar plate, is a selective and differential culture medium used in microbiology for the isolation and identification of Gram-negative bacteria, particularly members of the Enterobacteriaceae family, such as Escherichia coli and Salmonella species. The EBU plate contains eosin and methylene blue dyes, which selectively inhibit the growth of Gram-positive bacteria while allowing the growth of Gram-negative bacteria. Additionally, the medium differentiates between different types of Gram-negative bacteria based on their ability to ferment lactose. Lactose-fermenting bacteria, such as E. coli, appear as dark-centered colonies with a metallic green sheen, while non-lactose-fermenting bacteria, such as Salmonella, appear as colorless or pink colonies. The EBU plate is a valuable tool in clinical microbiology laboratories for the rapid identification and differentiation of Gram-negative pathogens, which is crucial for the diagnosis and treatment of various infectious diseases. Understanding the principles and applications of the EBU plate is essential for microbiologists and healthcare professionals working in the field of clinical microbiology and infectious disease management.