Biotechnology

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Revision Notes

Introduction & Basics of Biotechnology-

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1) What are some applications/uses of biotechnology?

Biotechnology is used in industrial/commercial applications, healthcare/medicine, agricultural/environmental, and forensics labs

Agricultural biotechnology helps to protect from insects/pests & to increase yields prevent damage from insects and pests and reduce farming’s impact on the environment

DNA fingerprinting is a common use of biotechnology to analyze evidence

Many drugs, such as insulin, or treatments, such as gene therapy, are made possible by

biotechnology


2) What was scientifically significant the 1970s that sparked the biotechnology era?

1. Discover and use of restriction enzymes & the ligase enzyme 

2. Cohen, Boyer, and Berg transferred recombinant DNA plasmid into a bacteria to produce an amphibian protein (1st case of genetic engineering)

3. Human insulin was synthesized in E. Coli using genetic engineering


3) What do the workplaces study?

Biotech companies- Work for a profit, to make commercial products

Forensics- Use forensic procedures (such as DNA fingerprinting) to analyze evidence

Environmental- . Environmental scientists may use similar DNA fingerprinting techniques to identify plant and animal breeding partners to control protected or endangered

species.

University/government research lab- “Pure science” research, do science for love of academia; to enrich scientific knowledge

University/government labs (CDC)- Conduct pure/applied science and use the results to further research or to provide information for the development of products to treat

diseases


4) Be able to recognize examples of genetic engineering.

GMO’s, synthesis of human insulin, anything genetically modified by humans


5) Understand how insulin was genetically engineered. Make sure to know the vocab associated with this process.

1. The gene for making insulin is cut from a length of human DNA using restriction

enzymes

2. It is inserted into a plasmid using ligase enzymes

3. The plasmid (now a vector) is placed inside a bacteria

4. The bacteria reproduces, resulting in millions of identical bacteria that produce human insulin


6) What is significant about the Human Genome Project?

Mapped entire human genome- helps us understand/identify genes & how gene expression works


7) What occurs during the research phase?

Using scientific data, scientists come up with an idea for a new product- they create a comprehensive development plan (mentioned below)


8) What occurs during the development phase?

Scientists now synthesize their product and undergo lab testing to prove that it works how it is supposed to- they demonstrate proof of concept (mentioned below)


10) Explain why testing doesn’t stop even after a product is released to consumers.

Side effects/issues with a product sometimes don’t appear for months or

years after a product is released- to ensure continued safety


11) What role does the FDA, EPA, and USDA have in the manufacturing of products?

FDA- Oversees the safety of most foods for humans and animals, including those

produced using biotechnology; the safety and effectiveness of intentional genomic

alterations in organisms produced using biotechnology, and in drugs produced using

biotechnology

EPA- Regulates biopesticides & certain microbial products of biotechnology (anything that

can harm the environment)

USDA- Protect agriculture from pests and diseases, ensure that biotechnology products

aren’t harmful to plant life


12) Explain the difference between a negative and positive control group.

Negative and positive control groups are used to demonstrate that the test is valid (specifically at time of use)

Positive control group- Gives predictable positive results

Negative control group- No exposure to experiment material; should test negative


13) When a company has a patent on a product, how long does it last? How is it beneficial for the company to patent their products?

Patents usually last 20 years and allow the company to make back the money they invested on their product as patents exclude others from making, using, or selling the

product.


Vocabulary:

Biotechnology-

the manipulation of organisms or their cellular components


Plasmid-

Small, circular DNA molecules found in bacteria


Restriction Enzymes-

Enzymes that splice DNA into fragments


Ligase Enzyme-

Like glue- used for pasting pieces of DNA together


recombinant DNA (rDNA)-

DNA produced by combining DNA from different sources


Vector-

rDNA is inserted into this to carry it


Genetic engineering

Modification/manipulation of an organism's genes using technology


Selective breeding/artificial selection-

Selecting organisms with specific traits to reproduce for more desirable traits


Genetically modified organisms-

organisms whose genes have been altered using genetic engineering


Genomics

Study of genes (structure, function, etc.)


Proteomics

Study of proteins (structure, function, etc.)


Comprehensive development plan-

Questions that a company asks about their product to determine if it is worth the initial investment: 

does the product meet a critical need?

Is the market large enough? 

Does preliminary data support that the product will work?

 Can patent protection be secured?

Can the company make a profit?


Proof of concept-

Using data collected from lab/animal tests it is determined if the product is feasible/works

how intended.

Is it possible to produce the new drug in sufficient amounts? What needs to be done to

ensure safety? Which characteristics are indicative of efficacy (proof that it is effective)?Is

it stable over time?


Product pipeline-

It takes many years (10-15) for products to complete the steps and this is referred to as being in the “pipeline” whereas many potential products are kept in the pipeline at once


Hypothesis-

It is NOT an educated guess

It is a testable statement that attempts to answer the research question


Experiment-

Procedure that tests the hypothesis- a valid experiment collects qualitative and quantitative data, uses control groups, tests a single variable, and undergoes multiple

trials


Conclusion – REE, PE, PA

REE- Results w/ evidence & explanation- discuss what happened and support with data

PE- Possible errors in the experiment

PA- Practical applications of lab results


Patent-

A form of intellectual property that gives its owner the legal right to exclude others from making, using, or selling an invention for a limited period of years.


positive control-

Control group that will test positive- one that will give predictable results


negative control-

Control group that is lacking what is being tested so as to give expected negative results- ensure test validity

Lab Safety-

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1. What does OSHA regulate?

Occupational Safety and Health Administration, regulates safety & work conditions for employees. OSHA ensures healthy working conditions for employees by setting and

enforcing standards and by providing training, outreach, education and assistance.


2. What does the NFPA system regulate?

The National Fire Protection Association is a marking system that uses a “safety diamond” to provide information on the health, flammability, reactivity and special hazards for many common chemicals.


3. What does the EPA regulate?

The Environmental Protection Agency (EPA) regulates the disposal of potential hazardous materials and the use of biotechnology products in the environment.


4. What was the significance of the ‘Incident in Bhopal, India’?=

The gas incident in Bhopal was caused by neglected safety conditions and an unaware community & response effort that resulted in many thousand lives lost. As such the US

(and other countries) passed increased safety rules & regulations (such as the EPCRA).


5. Identify the proper procedures for handling chemicals and biohazards.

1. All chemicals should be treated as hazardous until you learn otherwise

2. Label chemicals & read the label before opening

3. Wear appropriate PPE

4. Close containers fully & handle with care

5. Report spills/broken glassware

6. Use clean glassware

7. Dispose of chemicals properly

8. Clean everything thoroughly & wash hands when done (Do not put unused

materials back into the original container).


6. Identify the proper disposal of chemicals and hazardous materials.

● This process is regulated by the EPA

● All waste should be treated as hazardous, and no solutions should be washed down

the drain unless it is certain there is no health hazard

● Waste should go in specific waste containers

● Biohazards should be placed in an autoclave biohazard bag and sealed

● Glassware should be washed/autoclaved


7. What is PPE?

Personal protective equipment (PPE) is safety equipment that protects the user against specific hazards. The pictograms address the needs for additional PPE.

Safety glasses, gloves, lab coats and closed-toed shoes are standard lab PPE.


8. What is a MSDS and what sections are included on it?

Material Safety Data Sheet- Provides information on chemicals and includes the properties of each chemical.

1. Chemical identity - contains contacts for manufacturer and emergency.

2. Hazard ingredients/identity – components are listed.

3. Physical chemical characteristics – general properties such as how volatile it is, etc.

4. Fire and explosion hazard data

5. Reactivity data – reactivity patterns of the chemical.

6. Health hazards – toxicology data such as recommended emergency procedures.

7. Precautions for safe handling and use - how to deal with spills.

8. Control measures - recommendations for personal protective equipment (PPE).


9. What are the biosafety guidelines – BSL 1, BSL 2, BSL 3, BSL 4?

The Biological Safety Level (BSL) levels are guidelines pertaining to the handling of

biological organisms and molecules; classified by level of biohazard.

BSL 1- Minimum danger, microorganisms not known to cause disease (E. Coli, etc.)

BSL 2- Moderate danger, microorganisms are an infectious hazard (RSV, influenza)

BSL 3- Microorganisms that can cause serious disease (CoV-2 (Covid 19)

BSL 4- Exotic microorganisms that can be lethal (Ebola, Smallpox)


10. What is an SOP and why is it needed in a lab setting?

In order to ensure consistent results, a set procedure must be followed. A standard operating procedure (SOP) is a step-by-step outline for how to perform a particular lab

procedure or method.


11. What are GLPs and what are the requirements?

Some labs need to collect data based on good laboratory practice (GLP)– a quality system required for sound lab work. GLPs are used to develop test data on the properties and safety of chemicals, biological molecules, or organisms.

GLP requirements:

● Responsibilities & qualifications of all participants stated

● SOPs written/used for material acquisition and procedures done

● A detailed plan prior to the start of the study outlining the purpose, detailed test

methods, and how the results will be reported. Laboratory notebooks are an

integral part of how results are reported.

● The results and data generated by the study need to be properly stored and

archived.


Vocabulary:

Community Right To Know-

The provisions help increase the public's knowledge and access to information on

chemicals at facilities. Labs and industrial facilities must make it known to their

community what they work with and the subsequent risk.


EPCRA-

Based on the storage and handling of dangerous chemicals the Emergency Planning and

Community Right to Know Act helps communities with the planning and preparedness for

releases of chemicals.


autoclave biohazard bag-

Autoclaves use water, pressure, and heat to create superheated steam that sterilizes

objects. Waste should go in an autoclave biohazard bag made of a high melting point

plastic, sealed with autoclave tape, and autoclaved at high temperatures and pressures to

completely kill all organisms.

Solution Preparation-

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Accuracy & precision are terms that are used to describe the quality of measurement.


Accurate measurements denote that they are close to the actual/known value (close to the target).


Precision measurements are very close to each other.


Measurements can be accurate, precise, both, or neither. If a measurement is accurate & precise they are close to the true value, and do not differentiate from each other. A precise set of measurements might not be close to the true value, but will be close to each other! An accurate set of measurements might score close to the true value, but will not be close to each other. 


A solvent is the substance in which a solute dissolves (such as ethanol or water)

A solute is the substance that dissolves in a solvent


Mass is a measure of the amount of matter in an object. 


Molarity is the number of moles of a substance per liter of solution represented by “M”. (Moles meaning 6.02 * 1023 atoms of something). 


Concentration is the amount of substance present in a solution, usually measured in mass/volume, %mass/volume, or molarity.



The molecular/formula weight/molar mass is the atomic weight of all the elements in a substance, for example H2O contains two hydrogen atoms (at 1.01 each) and one oxygen atom (at 16.00) so the molar mass would be 18.


A concentration is usually known as the “stock solution” such as 10 M hydrochloric acid. If we diluted this by adding dH2O to get 1M hydrochloric acid, this new solution would be our “working solution.” 


Serial dilutions are continued dilutions of a stock solution based on a certain dilution factor, for example going from 1 M, 0.1 M, 0.01 M, 0.001 M, etc.




There are 4 main formulas for preparing solutions:


The first is with mass/volume where:

Concentration in g/mL * volume in mL = solute needed in g

If your values are given in values other than grams or militerers, you will need to convert. Additionally, if you are given a concentration in percent, move the decimal point two places left of the number. 


For example if we had magnesium sulfate (MgSO4) and we wanted to make 200mL of a 15 g/mL solution we set it up as 15 g/mL* 200 mL = 3000g MgSO4 needed. 

To make the problem slightly more difficult if we needed .1L of 20mg/mL of MgSO4 solution we need to convert and get .02 g/mL * 100 mL = 2g MgSO4 needed.


To solve for variables on the left side of the equal sign:

If we wanted to know this concentration of 40g phosphorus pentoxide (P4O10) in 400mL of distilled water we’d set up and solve the equation to get a 0.1 g/mL concentration.

(We would do the same process to solve for volume)

x * 400 = 40g → 400x400=40400

The next method for calculating concentration is by using molarity. The molarity formula is slightly more complicated as we require a molar mass (see vocab) and as we use a different unit (M) where:

Volume in L * Molarity * Molar Mass = Solute needed in g

You will need to use a periodic table to add up the atomic weight of all the elements in the compound given for your molar mass. Additionally, since molarity is measured in mol/liter you must make sure your volume is in liters! 


For example if we wanted 1L of 1.0M magnesium hydroxide (MgOH2) we set it up as 1 * 1 * 42 = 42g MgOH2 needed.

The 42 is the result of adding the atomic weight of magnesium (24), oxygen (16), and hydrogen (1 * 2) together.


To solve for volume:

If we wanted to know how much solvent to add to a mixture of 34g of 2.0M barium nitrate (BaNO3) solution we would set up the equation like so:

x  * 2 * 199 = 34 → 398x398=34398

We end up with 0.0854 liters, which we can convert to 85.4 mL to make it more manageable.



We can also dilute a pre-made stock solution using the formula C1 * V1 = C2 * V2  where C and V stand for concentration and volume, respectively. 

If we had 5M sulfuric acid (H2SO4) and we needed 50 milliliters of 0.1M sulfuric acid, we would need to dilute our stock solution. 

We start with our stock concentration for C1 with V1 being our unknown value. Then we plug in 0.1 and 50 for C2 and V2 to get

5 * x = 0.1 * 50 → 5x5=55

We end up needing 1 mL of stock solution out of our total 50 mL meaning we need 49 mL of H2O. 


The final form of solution math is a proportional formula where we can use a given proportion to calculate the amount of substance needed using M1V1=M2V2 where M and V stand for mass and volume, respectively. 

For example if instructions to prepare sodium alginate included mixing 30 grams of powder with 60 mL of water and we needed to make 500 mL of sodium alginate the equation would be

30/60=x/500where we can then cross-multiply and solve to get 250g needed.

Practice:


How many grams of magnesium oxide (MgO) are needed to make a 40 mg/mL in 100 mL solution?



How many grams of sodium chloride (NaCl) are needed to make a 10 g/mL in 50 mL solution?



What is the concentration of 60g CuSO4 in a 120 mL solution?



How much rubidium chloride (RbCl- 121 g/mol) is needed to make 2 liters of a 0.1 M solution?



How much potassium permanganate (KMnO4- 158 g/mol) is needed to make 500mL of a 1M solution?




How much dH2O do you need for a solution of 25g potassium chloride (KCl) in a 0.5M solution?



If you needed 200mL of 50% methanol from a 90% stock solution how much methanol and dH2O would you need?



If a 1mL of a 40X solution were diluted down to 20mL final volume, what would the new concentration be?



How would you dilute 8M sodium hydroxide to make 160mL of a 0.1M working solution?












Scientific notation is a system that, simply, tells you how to move the decimal point in a number. It is a shorthand notation for large or small numbers. Negative exponents tell us to move the decimal left and positive tell us to move it right. 

If we had a number like 3.07 * 105 we would move the decimal right 5 times to get 307000. However, if we had 3.07 * 10-5 we would move the decimal left 5 times to get 0.0000307. If we start with a number that is not in scientific notation we move the decimal to right before the first number, then write the exponent correctly for the movement direction.


Micropipettes 


Micropipettes are precision instruments used to measure exact amounts of liquids. When using a micropipette select the one with the smallest range to suit your needs to increase precision. 


To withdraw a liquid:

-Set the volume

-Press micropipette onto tip

-Depress plunger to the first stop

-Insert into liquid 

-Slowly release plunger

-Pause before lifting pipette


To dispense a liquid:

-Insert into delivery tube

-Depress plunger to the second stop

-Pause

-Lift tip out of tube

-Release plunger slowly

-Eject tip

Lab Techniques & Safety-

Cellular & Microbiology-

Cell Cultures-

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Bacterial Transfer

Agar Prep

Koch’s Postulates

Aseptic Techniques

Vocabulary


Genetic Engineering- 

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Vocabulary:

genetic engineering- manipulation of genetic information

multiple cloning sites- series of unique restriction enzyme recognition sites

vectors- Vessel to carry genetic information with (often a plasmid)

lysosome- degrades peptidoglycan in cell walls

precipitant- molecular debris, found at bottom of test tube

supernatant- suspends DNA, found at top of test tube

lysed- to explode cells

detergent- Solutions to lyse cells with

protease- Degrades proteins

salts- clumps cellular debris

probes- single-strands of DNA or RNA that bind to a complementary sequence that have a biological marker attached. Shows location of DNA sequences.

hybridization- Process where probe binds to complementary sequence

restriction enzymes- cuts DNA at specific sites

gel electrophoresis- separates DNA fragments based on size

DNA polymerase- Synthesizes new DNA strands in the PCR process

thermal cycler- Used in PCR to take DNA through temperatures best for replication (95°, 50°, 72°)

polymerase chain reaction- used to replicate specific sequence of DNA many times

southern blotting- transfers DNA from gel onto membrane with probes. Uses photographic film to identify specific sequences of DNA

exons- coding sequences that leave the nucleus

introns- non-coding sequences of DNA that cannot leave the nucleus


Flashcards

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