Bacterial Transformation

Bacterial Transformation. Chapter 5: Background. era rans orma 10 and Plasmid Purificatio.

[Audio] The objective for this chapter is to understand the basic features of a plasmid and their importance in genetics. A plasmid is an extrachromosomal circular DNA molecule that is double stranded and typically found in bacteria. It is capable of replicating independently of the chromosomal DNA and can be transferred from one bacterium to another. Plasmids can carry genes that confer an advantage on the bacteria they reside in, including antibiotic resistance. Plasmids can be artificially created using recombinant DNA technology and be used in many biotechnological applications..

[Audio] Plasmids were first discovered by Joshua Lederberg and William Hayes in the 1950s as extragenomic loops of DNA. They are now used by scientists to engineer and express genes in bacteria, as well as produce proteins for medical and research applications. For example, the gene coding for human insulin was inserted into a plasmid in 1978 and expressed in bacteria, allowing them to produce human insulin which has become critical for medical research and treatments. Additionally, the green fluorescent protein is used to study the function of genes. Essentially, plasmids are the DNA that is transferred from one bacteria to another, providing scientists with an efficient way to further investigate the field of genetics..

[Audio] Plasmids are found in many bacteria, being small, independent loops of DNA that are not part of the bacterial chromosome, but existing independently and self-replicating within the bacterial cytosol. They have an origin of replication, or ori - a particular DNA sequence - that enables them to be replicated. Plasmids bring their host bacteria a range of benefits, such as antibiotic resistance, new enzymatic activities and the capacity to absorb and utilize fresh metabolic pathways..

[Audio] In this chapter, we will be discussing the process of bacterial transformation. This process, which is used in genetic engineering, is used to incorporate foreign DNA fragments into the bacterial genome. We will discuss what happens during the process of transformation, and how transformed bacterial cells can be identified. Let's begin by discussing the background of bacterial transformation..

[Audio] Transformation is the process of introducing a plasmid into a cell. A plasmid is a small, circular piece of DNA that can exist apart from the main chromosome, and has the ability to replicate independently. To perform transformation, a special strain of bacteria, called competent E. coli, is treated to make it receptive to foreign DNA, like the plasmid. After treatment, the bacteria is capable of taking up the plasmid, thus allowing scientists to introduce new traits, which can be used in research and production..

[Audio] Bacterial transformation is a rare occurrence, with a ratio of one in every hundred to one million cells taking up the plasmid. Despite the majority of the cells in the bacterial lawn not having undergone transformation, we must still discover the few that did. This slide shows the difficulty in locating those transformed cells, and then we will analyze the procedures and techniques used to identify them..

[Audio] A necessary process when attempting to cultivate new bacterial or yeast cells for testing or other purposes is the selection of transformed cells. Transformed cells obtain new traits due to the insertion of novel genetic information. The selection of these cells can be done by the utilization of agar plates with selective media, usually incubated at 37°C overnight. Once the plates are incubated and ready, the transformed cells can then be identified, thus enabling the continuation of the research..

[Audio] Non-selective media allow all types of cells to grow, while selective media contains substances that allow only transformed cells to grow. Selective media can be used to select for transformed cells, as in the laboratory. An example is the LB/Amp plate containing the antibiotic ampicillin, used to select for cells transformed with a plasmid containing the ampicillin resistance gene..

ddO mod we u. Antibiotic selection. Transformed bacteria contain the plasmid.

[Audio] Beta-lactamase is an enzyme that breaks down beta-lactam antibiotics such as penicillin and cephalosporin, naturally occurring in some bacteria, but also artificially produced in a laboratory. Its use has created resistant bacterial strains, useful for studying antibiotics and drug resistance. A comparison between a bacterial strain with beta-lactamase (on the left) and one without (on the right) demonstrates the strain on the left appearing larger and more visibly transformed, likely due to its increased ability to survive in the presence of the antibiotic..

[Audio] Ampicillin, often referred to as Amp, is a type of antibiotic that is commonly used to treat bacterial infections. It is effective because when added to food, it selectively targets bacterial cells and leaves healthy cells alone. Consequently, Ampicillin is beneficial for distinguishing which cells should be targeted for treatment and can be useful for controlling infections. In short, it can be thought of as selecting the candy from the bowl that you want to eat..

[Audio] Transformation efficiency in molecular biology is the number of new transformed cells divided by the number of DNA molecules that were introduced into the cells. To calculate transformation efficiency, one must multiply the number of colonies or transformed cells by the volume of cells and divide it by the amount of DNA added. Understanding transformation efficiency is essential in determining the success of gene transformation when dealing with gene cloning in molecular biology..

[Audio] Measuring transformation efficiency is key for cloning genes and for other uses. This is done by determining how many colony forming units (CFU) are produced per microgram of plasmid DNA in the transformation process. The number of CFUs is used as the metric to gauge the transformation efficiency..

[Audio] The equation can be used to determine the amount of plasmid that ended up on the agar plate. Rearranging the equation shows that 10 μl of the transformed volume contains 50 ng of plasmid. Since 60 CFU are present on the plate, it can be deduced that each CFU holds 0.833 ng of plasmid DNA..

[Audio] Divide the CFU (colony forming units) by the amount of plasmid spread on the plate (0.001 pg) to obtain the transformation efficiency, which would be 60000 CFU/pg. The total volume of the transformation was 10 pl times 0.05 ug or 500 pl, meaning there was 0.001 pg of DNA spread on the plate. In order to calculate transformation efficiency, the volume spread of the DNA and the amount of DNA in the transformation must be known..

[Audio] This chapter will look into the significance of each step in a transformation procedure. We will explore what each step entails and its significance for attaining the desired outcome. Furthermore, we will recognize the importance of adhering to the steps in their precise order and how it can influence the outcomes. With this discussion, you will gain a greater understanding of the transformation procedure and how best to use it to accomplish success..

[Audio] This slide illustrates the transformation method used to introduce plasmids to bacteria. The transformation process relies on the ability of calcium chloride to create pores in the bacterial cell wall, allowing the plasmids to pass through. The process begins by mixing the plasmids and bacteria in the presence of calcium chloride. As the concentrations of plasmids and bacteria increase, the calcium chloride induces the transformation of the bacteria. This process is used to introduce genes or other genetic material to the bacteria, allowing them to be expressed in yet another organism..

[Audio] Calcium Chloride, commonly known as CaCl2, is a compound of calcium and chlorine. It is an important element in many industrial and manufacturing processes, as it helps to neutralize the negative charge on DNA molecules. This helps to facilitate the replication of DNA, and is an essential part of the molecular structure of cells. By neutralizing the negative charge, calcium chloride helps to maintain the integrity of the DNA molecule, ensuring that genetic material is replicated accurately. It is also used in a variety of other industrial applications, including as a food preservative, a pH-balancer for swimming pools, a softening agent for water, and a road de-icer..

[Audio] The process of transformation involves several steps. First, a mixture containing bacteria and plasmids is incubated with LB Broth, which provides the necessary time for the beta-lactamase enzyme to be made. Next, the mixture is heat shocked at a temperature of 42ºC, allowing the plasmids to enter the cells. Afterwards, the Calcium Chloride transformation method is used, as the plasmids are mixed with the bacteria in the presence of Calcium Chloride (CaCl2). Finally, the mixture is placed on ice, slowing the fluidity of the cell membrane. With this method, the transformation process is complete. Thank you for listening..