Genetic Engineering Principles

The manipulation of genetic information with the goal of producing organisms with new or improved characteristics.

1. Isolate the DNA for the desired characteristic/protein from a donor cell.
2. Insert the isolated DNA into a vector (rDNA).
3. Introduce the vector into host cells to produce recombinant cells.
4. Grow recombinant cells in culture, first on a small scale (fermentation) and then on a large scale (manufacturing) to produce the desired protein product.
5. Isolate and purify the recombinant protein product from the cell culture.
6. Analyze the protein for activity and other characteristics before releasing it to the market.

The first step in retrieving DNA from a cell is always to break open, or lyse, the cells. This ensures the DNA is separated from the rest of the cell's contents.

A procedure that requires a sufficient quantity of starting cells grown in a broth or agar culture. It involves breaking open the cells by using lysozyme, detergent, and other enzymes to release the bacterial chromosome. The DNA is then separated from cell debris by centrifugation.

The enzyme that degrades bacterial cell walls by decomposing the carbohydrate peptidoglycan.

The liquid that remains after a precipitate has been spun down to the bottom of a vessel by centrifugation.

Similar to extracting bacterial chromosomal DNA, but with the addition of alkaline lysis solution to degrade and precipitate the cell walls, protein contaminants, and bacterial chromosome.

The process of searching for a specific gene within purified DNA. It involves using a probe, a DNA/RNA molecule complementary to the target sequence. Techniques like restriction enzymes, gel electrophoresis, and Southern blotting are used to identify the gene.

A method of probing that involves cutting DNA with restriction enzymes, separating the fragments by length on a gel, and then applying labeled probes to identify the gene of interest.

A technique that uses primers to replicate millions of copies of a specific DNA sequence within a few hours.

Small strands of DNA that act as starting points for DNA synthesis or replication.

A DNA copy synthesized from mRNA using reverse transcriptase. It is complementary to the original DNA sequence of interest.

An instrument used for PCR reactions that automatically cycles through temperature changes to facilitate DNA replication.

Once the gene of interest is isolated, it can be transferred into new host cells like bacteria or yeast. This process is called genetic transformation, which allows the host cells to express the new gene and produce a non-native protein. This ability has fueled the development of the biotechnology industry.

The uptake and expression of foreign DNA by a cell.

One of the first transformations involved transferring the human insulin gene into E. coli bacteria. This enabled bacteria to produce human insulin, revolutionizing diabetes treatment.

Bacterial DNA that is easily taken up by some bacteria cells and can be used as vectors to carry genes of interest into cells.

Using viral DNA as vectors to deliver genes of interest into cells. This is called transduction, and it is often used to transform mammalian cells. It involves inserting the gene into the viral DNA using restriction enzymes.

The process of transforming mammalian cells with new genetic material. It is very similar to bacterial transformation but has different techniques, with specific types of mammalian cells being used as host cells.

Enzymes that cut RNA or DNA at specific sites. Restriction enzymes are endonucleases that cut DNA at specific recognition sequences. This helps isolate DNA fragments for various purposes, especially when creating recombinant DNA.

The restriction fragments produced by endonucleases that have one end of the double-stranded DNA longer than the other. This is crucial for joining DNA fragments from different sources to create recombinant DNA.

The process of creating recombinant DNA, the joining of DNA fragments from different sources. Requires two main enzymes: restriction enzymes to cut DNA at specific sites and DNA ligase to paste the fragments together. Recombinant DNA is like a hybrid DNA molecule.

A plasmid vector is opened by a specific restriction enzyme. That same restriction enzyme cuts the gene of interest leaving each strand of DNA with matching sticky ends. The enzyme DNA ligase then seals the pieces of DNA with matching sticky ends, creating recombinant DNA. The rDNA, now containing the gene for the desired protein, is inserted into a bacteria cell. The transformed cell then expresses the newly acquired DNA by converting it into mRNA and then creating the protein from it.

The pieces of DNA that result from a restriction enzyme digestion. They are used to cut DNA into manageable pieces for analysis and can be used for DNA fingerprinting, evolutionary studies, and to understand DNA mutations.

Restriction fragment length polymorphisms, or RFLPs, are different lengths of DNA fragments produced from restriction enzyme digestion due to variations in the genetic code for the same gene between individuals. RFLPs can be detected by gel electrophoresis.

Applications of RFLP analysis include DNA fingerprinting, evolutionary studies, and understanding DNA mutations that lead to differences in species. RFLP analysis helps scientists see differences in DNA sequences based on the length of the resulting fragments.

Determining the order of restriction sites of enzymes in relation to each other. This method was primarily used to sequence plasmids and other small DNA sections.

A process involving 6 steps:

1. Grow host cells in broth culture.
2. Make cells competent for DNA uptake using CaCl₂ or MgCl₂.
3. Add the rDNA plasmids to the cells.
4. Heat shock cells to aid in DNA uptake.
5. Add nutrient broth for cell recovery and gene expression.
6. Plate cells on selection media.

The ability of a cell to take up DNA, which is increased by treatment with CaCl₂ or MgCl₂.

The process of separating and identifying transformed cells from a mixture by growing them on agar plates containing a selection media that allows only transformed cells to survive and grow.

In a laboratory setting, the process of transforming cells is often inefficient. To increase efficiency, scientists create large pores in the cell membrane, allowing for easier DNA entry. They also use techniques like heat and cold shocks to increase DNA uptake.

A measure of how successfully cells have acquired new genetic material, which indicates the efficiency of the transformation process.

After the transformation process, cells are given nutrients and a recovery period to repair their membranes and express their newly acquired genes. Cells that survive this process are able to grow and divide on a selection media.

A gene that codes for the enzyme beta-galactosidase, which converts the carbohydrate X-gal into a blue product. It is often used as a marker gene in genetic engineering experiments.

The systematic increase in the size or volume of production of a particular product. It involves scaling-up the culture from small volumes to larger volumes like spinner flasks and fermenters, while monitoring cell growth and product yield.

A type of flask commonly used for scale-up in which there is a spinner apparatus (propeller blade) inside to keep cells suspended and aerated.

Automated containers used for fermentation. These are designed to be easily monitored and controlled for growing microorganisms and producing bio-engineered products.

Assays are used during the scale-up process to monitor and verify the protein of interest. The Assay Services department develops and conducts testing for the quality and activity of the product. Another department, Quality Control (QC), performs additional testing and verification to ensure the product meets quality standards.

A form of selection used to identify transformed cells. This method involves adding an antibiotic-resistant gene to the rDNA plasmid, ensuring only the transformed cells with the plasmid can grow on the selective media.

The process of breaking open cells to release their DNA. It's the first step in DNA isolation and involves using various enzymes and solutions to degrade cell membranes and release the genetic material.

A piece of DNA created by combining DNA fragments from different sources. Often a plasmid is engineered to carry the gene of interest.

A special type of DNA molecule used as a vehicle to carry foreign genes into cells. It's often a circular piece of DNA found in bacteria.

A treatment used to increase the efficiency of DNA uptake by cells during transformation. This involves using specific salts like calcium chloride or magnesium chloride to make the cell membrane more permeable to DNA.

A method that involves treating cells with heat and cold shocks to make their membranes more permeable to DNA uptake. This is a common technique used in genetic engineering experiments.

The process of growing transformed cells in controlled environments to produce the desired protein product. It typically involves scaling up cell cultures from small volumes to larger volumes, often in fermenters.

These are procedures carried out during the scale-up process to monitor cell growth and assess the expression of the gene of interest. They ensure the product is being produced correctly and meets quality standards.

The final phase of cell growth where the rate of cell division slows down due to limited resources or waste accumulation.

The use of restriction enzymes to cut DNA into fragments of different sizes. It can help to identify the location of specific genes and can be used for DNA fingerprinting and genetic studies.

A specific DNA sequence recognized and cut by a restriction enzyme. It acts as a target site for the enzyme, allowing for precise cutting of DNA.

The chemical process that seals the gaps between DNA fragments after they have been cut by restriction enzymes. It's an essential tool in recombinant DNA technology.

The process of extracting DNA from bacterial cells. It involves using specific solutions and methods to break open the cells and purify the DNA.

A type of DNA that is synthesized from RNA using the enzyme reverse transcriptase. It represents a copy of the original mRNA sequence, providing a DNA version of the gene.

The stage of cell growth where cells are actively dividing and expanding, leading to a rapid increase in cell population.

The process of identifying, isolating, and confirming that the correct DNA segment has been inserted into a vector. This is a critical step before the vector can be used to transform cells.

A test used to determine the presence and activity of a specific protein. It's used to confirm that the transformed cells are successfully producing the desired protein.

The use of alcohol (ethanol) to precipitate and collect DNA from a solution. This is a common technique used in DNA isolation and purification.

The combination of a DNA fragment that contains the gene of interest and a vector that can carry and deliver the DNA to host cells.

A series of steps involved in transferring the desired gene into host cells. This includes DNA ligation, bacterial transformation, and verification of the transformed cells.