Biotech for hosa

Biotechnology

The use of modern molecular and microbial techniques to make useful products or processes.

Vocabulary/Terms

Adherent Cultures

So cultures that adhere or stick to a solid surface, such as tissue culture, dish, or flask

Insulin

A hormone regulating blood sugar and commonly used to treat diabetes.

Affinity

An attraction or likeness to something

Enzymes

Biological catalysts, speeding up chemical reactions in living organisms

Chromatography

A technique used to separate and analyze complex mixtures based on their differential interaction with a stationary phase and a mobile phase.

Polyadenylation

The addition of a poly(A) tail to the 3' end of eukaryotic mRNA, playing a role in mRNA stability and translation.

Release Factor

A protein that binds to the ribosome when a stop codon is encountered, leading to the termination of protein synthesis.

DNA Polymerase

Enzyme used in PCR to extend the primers by adding complementary nucleotides to the template DNA, usually from thermophilic bacteria

Fluorescence

The emission of light by a substance that has absorbed light or other electromagnetic radiation.

Chapter 1

The Biotechnology Industry

Genetic Engineering

Process of manipulating genes

Genomics

Investigates whole genome, full complement of DNA in cell

Proteomics

Studies proteome, entire protein complement of cell

Metabolomics

study of metabolic expression in cells

Microbiomics

Investigates microbiome, all microorg. living in a place at the same time

Bioinformatics

Info tech for biology applications. Gather, store, sort and analyze data.

Biosensors

Biotech instrument that converts biological actions of molecules or organisms to eletricsl signals.

Bioremediation

Using organisms to convert hazardous waste into less hazardous. Organisms that clean up waste are naturally occurring or genetically engineered.

Endangered animals

Species at risk of extinction due to factors such as poaching, habitat loss, and environmental changes.

Innocence Project

Non-profit organization using DNA evidence to exonerate wrongfully convicted individuals.

Genetic variability

Differences in DNA sequences among individuals, used for identification and tracing ancestry.

Genographic Project

Initiative mapping human migration patterns through DNA analysis.

DNA profiling

Process of analyzing and identifying individuals based on unique DNA patterns.

Nanobiotechnology

Application of biological molecules at atomic and molecular scales for specific functions

Rexin-G

Nanotechnology-based drug targeting metastasized pancreatic cancer cells.

Aquaporin

Protein utilized in artificial membranes for purifying water on a nanoscale.

Biotechnology Industry Worth

Expected value to exceed $700 billion by 2025, reflecting its significant economic impact.

Start-Up Biotechnology Company

Emerging company with innovative teams, often backed by private funding.

Chapter 2

Laboratory Safety

General Product Development Timeline

Conceptualization, Research, Development, Commercialization

Conceptualization

Market research finds need for product, researchers confirms product can be made. Legal work as well.

Research

Scientist create initial products, depends on type of product

Development

Refine product, investigate best manufacturers, detail specifications, testing (safety/effciency)

Commercialization

Details of marketing manufacturing and distribution decide, preparation for sale

Orphan Drug Act (ODA)

Incentivizes pharmaceutical companies to develop drugs for rare diseases, which are those affecting fewer than 200,000 people in the United States.

Quality Assurance Department

Ensures that the company complied with its own quality system and any applicable government regulations. (Requires records be maintained on operations). Makes business dealings smooth and other companies more likely to work with you.

Good Laboratory Practice

A quality system, a set of regulated practices used to collect safety data on product during development

What does GMP stand for?

Good Manufacturing Practices

What is the purpose of Good Manufacturing Practices?

To ensure steps are taken to produce consistently safe and effective products.

Return on Investment (ROI)

Financial metric evaluating the gain from an investment relative to its cost, crucial for biotechnology ventures

Patent

Exclusive rights granted for a new invention, crucial for protecting biotechnological innovations.

Food & Drug Administration (FDA)

Regulatory agency ensuring safety of food, beverages, and therapeutic drugs, vital for biotechnology products

Environmental Protection Agency (EPA)

Regulatory agency safeguarding human health and the environment from pollutants and toxic substances, impacting biotechnology practices.

United States Department of Agriculture (USDA)

Regulatory agency ensuring protection for agriculture, livestock, and genetically modified organisms, relevant to biotechnology.

Occupational Safety and Health Administration (OSHA)

Regulatory agency ensuring workplace safety and health for employees, crucial for biotechnology companies

National Bioengineered Food Disclosure Law

Legislation requiring guidelines for labeling foods containing genetically modified organisms (GMOs), impacting biotechnology and consumer awareness.

Non-GMO Project

Not-for-profit agency providing third-party verification and labeling for foods not containing GMOs, relevant to biotechnology and consumer preferences.

Animal and Plant Health Inspection Service (APHIS)

Regulatory agency overseeing field testing of genetically modified plants and their impact on agriculture and the environment, crucial for biotechnology and environmental impact assessment

Chapter 3

Microbiology and Cell Culture

What is a single colony?

A colony from a single bacterium that has multiplied on a solid medium. It's like a round visible dot.

What is the purpose of the quadrant method?

Allows isolation of a single colony from a bacterial culture by splitting the plate into quadrants and diluting the bacterium repeatedly as loop a streak through each quadrant.

How do serial dilutions allow you to count bacteria?

Serial dilutions allow the number of bacteria to be diluted by tenfold (normally). There will be much less bacterium in the solution, allowing for accountable size of CFU's on the plate.

What wavelength of light do you set a spectrophotometer to if you want to detect bacteria?

600nm

In gram staining which is the decolorizer?

Alcohol

In grim staining what is the color of each stain?

Crystal violet is purple and Safranin is pink

Is a bacteria is gram positive

It has a think peptidoglycan layer

If a bacteria is gram negative

It has a thin peptidoglycan layer

Which type of bacteria is more resistant to antibiotics? Why?

Gram-negative because their unique cell wall structure. Gram-negative bacteria have a thinner peptidoglycan layer and an outer membrane, which act as a barrier to many antibiotics. This outer membrane contains pores and can actively expel antibiotics, reducing their effectiveness.

What color is gram positive bacteria?

purple

What color is gram negative bacteria?

pink

Chapter 4

DNA Structure and Analysis

Recombinant DNA

DNA that has been assembled by linking fragments of DNA from different organisms

Restriction Enzymes

Bacterial enzymes that cut DNA in very predictable locations

What are bacteriophages?

Viruses that inject their DNA into bacteria and then use the bacterial cellular machinery to reproduce more copies of themselves.

How did restriction enzymes evolve as a defense against bacteriophages?

Restriction enzymes evolved to cut the DNA that was inserted by bacteriophages from the bacterial DNA destroying the phage.

How do bacteria protect their own prokaryotic DNA from being lysed restriction enzymes?

The phosphate on the 5' and of the plasma DNA is removed using an enzyme phosphatase.

What does it mean that the restriction sites tend to be palindromic?

It means that the sites cut from 5' to 3' read the same from 3' to 5'.

Blunt End

Sticky End

After one cut, how many pieces does a plasmid have?

One piece

How many cuts/ restriction site does there need to be to divide the plasmid into two pieces.

Two cuts

Ligase

An enzyme that reforms phosphodiester bonds.

Why might it be easier to connect sticky ends than blunt ends (assuming the sticky ends were cut with the same restriction enzyme)?

The overhang from single each strand would have the complementary stares of the overhang from the other strands. The hydrogen bonds between the two complementary strands would allow for easier joining.

What are plasmids?

small extra genomic, circular loops of DNA, found in bacteria

Why is there a higher probability of the plasma ligating to itself then incorporating a new fragment?

The two ends of the open plasmid are in close proximity to each other while the DNA fragments are floating around the solution.

How do we stop the DNA from ligating to itself, and allow it to incorporate a new fragment?

The phosphate on the 5' end of the plasma DNA is removed using an enzyme phosphatase. The DNA can only recirculate if the new fragment is added.

Why are reaction buffers supplied with enzymes?

It ensures that the enzymes are provided with the optimal reaction conditions.

What is double digest?

When two restriction enzymes are used to digest DNA the same tube.

What is commonly in a restriction enzyme buffer?

The optimal salts and pH, for that specific enzyme, some also require additives such as bovine serum albumin.(BSA)

Why doe enzymes come in glycerol?

Enzyme in glycerol will not freeze at -20C (recommended storing temp). Repeated freeze-thawing of enzyme should be avoided since it reduces enzyme activity.

Where should enzymes stay on when actively working in the lab?

Ice or at 4C

What substances are included in a typical restriction digest?

Template DNA, Restriction Enzymes, Buffer, BSA(Optional)

What does CRISPR stand for?

Clustered Regularly Interspace Short Palindromic Repeats

What were all of those sections of spacer DNA in between palindromic repeats likely from?

Viruses/ Bacteriophages when a bacterium or archaeon is infected by a virus, the microbe captures some of the viral DNA and it sorted it into a CRISPR sequence as a space.

How will CRISPR spaces be perfect for hybridizing to the viral DNA if it ever shows up again?

Since the crisper sequence is contained in genomic DNA and is passed on to each generation and the library continues to grow. Should a matching virus infect daughter cells descended from this bacterium the spacer RNA transcript and CAs 9 complex would bind and cleave to the viral DNA to prevent it from replicating.

Cas 1 and Cas 2

Cut and capture the viral DNA.

The second phase of taking in viral DNA

The spaces are transcribed in each binds to a Cas 9 protein to form a "search and destroy complex" in the cell.

When introducing new genes by transformation, the genes may insert themselves in a way that disrupts other genes affect their gene expression or cause other unintended effects. This is assuming that a genius directly being placed inside genome (not as a plasmid). Explain where in a gnome, whether encoding or non-coding regions a gene could be placed that might have such impacts.

• Could be placed in the middle of a gene

-A placing between nucleotides Between that alter the frame reading for codons.

What makes CRISPER-Cas9 technology so beneficial?

Allow scientist to manipulate genes and gene expression, precisely, and in the least intrusive manner possible.

Cas 9 enzyme

Endonuclease that cuts double strained DNA at a site dictated by the particular God RNA that is bound to the Cas 9

Guide RNA (gRNA)

RNA approximately 100 nucleotides long that form a complex with CAs 9. A 20 nucleotide region at the end of the gRNA contains a spacer sequence complementary to the target DNA sequence.

How does CRISPR utilize cellular machinery?

The cell recognizes the DNA is damaged and

starts to repair it. Scientist take advantage of the ways that cellular DNA repair machinery works to introduce changes to one or more genes.

Using CRISPR on somatic/body cells only affects the individuals who you do it on. Why might people find it ethically questionable to use gametes or germ line cells?

With gametes the CRISPR mutation would get passed on to that organisms or persons offspring.

Why are combs placed on the side closest to the negative electrode?

DNA fragments are negatively charged. They repel from the negative electrodes and are attracted to the positive.

What are the most common electrophoresis buffers?

Tris/acetic acid/EDTA (TAE) & Tris/boric acid/EDTA (TBE)

What are DNA size standards?

Sample that contains DNA fragments of known sizes for comparison of unknown fragments.