Bse 2004 Exam 2

Front 1

The First Law of Thermodynamics

Back 1

"Energy cannot be created or destroyed."

It can only change from one form to another.

Front 2

Mechanical Energy

Back 2

Referred to as "work"

KE = 1/2 mv^2

m = mass

v = velocity

Front 3

Thermal Energy

Back 3

Referred to as "heat"

Front 4

MAEB Lab: The system that is being described

Back 4

Where we have hot andcold water entering the system and warm water leaving the system.

Front 5

The Mass Balance of the described system:

Back 5

Front 6

The Energy Balance of the described system:

Back 6

Front 7

DNA Lab: Step1 of the lab procedure says: “pipette 1 ml of the culture into amicrocentrifuge tube”. What is “the culture”?

Back 7

“Theculture” is a liquid bacterium that was used to purify the plasmid.

Front 8

DNA Lab: Step2 of the lab procedure centrifuges the sample. Is the DNA in the liquid orsolid?

Back 8

TheDNA is in the solid.

Front 9

DNA Lab: What does solution I do?

Back 9

The Re-suspension solution containing Tris, EDTA, glucose,and RNase. Glucose maintains the osmotic pressure so the cells don’tburst and RNase A is included to degrade cellular RNA when the cells are lysed.

Front 10

DNA Lab: Afteradding solution I and shaking or flicking, is the DNA in a liquid or a solid?

Back 10

After adding solution shaking, and I the DNA is in a liquid.

Front 11

DNA Lab: What does solution II do?

Back 11

The alkaline lysis solution contains NaOH, which breaks down the cell wall, andSDS, which breaks down the cell membrane and abolishes the proteins in the cell. And DNA to strand.

Front 12

DNA Lab: After adding solution II and gently mixing the solution, is the DNA in a liquid or solid?

Back 12

Proteins and junk are solid DNA dissolved

Front 13

DNA Lab: What does solution III do?

Back 13

Solution III contains potassium, which neutralizes the pH.

Front 14

DNA Lab: Why does the sample become cloudy?

Back 14

The solution becomes cloudy due to the insoluble cell fragments in the liquid.

Front 15

DNA Lab: Step 10 of the lab procedure centrifuges the sample for 10 minutes. Why is it important to avoid pipetting any solid to the spin column?

Back 15

It is important to avoid pipetting any solid to the spin column because the solid contains only debris and waste that could taint the solution.

Front 16

DNA Lab: How would you explain to a stranger what a “spin column” is?

Back 16

A spin column contains a silicon membrane that binds theDNA.

Front 17

DNA Lab: What is the purpose of the spin column in the procedure?

Back 17

The purpose of the spin column in the procedure is to purify the DNA.

Front 18

DNA Lab: Insteps 15-18, the column is “washed”. Where is the DNA during the wash?

Back 18

The DNA is located in the spin column membrane.

Front 19

DNA Lab: In step 22, an elution buffer is added to the solution. What does the elution buffer do?

Back 19

The elution buffer removes the DNA from the filter, and the DNA is collected.

Front 20

DNA Lab: After the sample is centrifuged in step 24, where is the DNA?

Back 20

The DNA is now in its purified form.

Front 21

DNA Lab: What was your DNA concentration?

Back 21

The DNA has a concentration of 31.5 ng/uL liquid.

Front 22

DNA Lab: What is a DNA ladder?

Back 22

A DNA ladder is a set of DNA fragment values that are in separated bands in a gel matrix in the shape of a ladder.

Front 23

DNA Lab: Why is a DNA ladder used in gel electrophoresis?

Back 23

The DNA ladder is used in gel electrophoresis to monitor the DNA migration for linear DNA fragments as a function of their length.

Front 24

DNA Lab: When working with live cultures, like E. Coli.,where should waste NEVER go?

Back 24

When working with live cultures, like E. Coli,waste should NEVER go in the trash or a sink.

Front 25

Glucoamylase (GA)

Back 25

-An ubiquitous enzyme among all vertebrate,plants, and diverse microorganisms.

- It catalyzes the production of glucose from the non-reducing end of starch and maltooligosacchar­ides.

Front 26

What is an Enzyme?

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A protein that can catalyze a specific reaction

Front 27

EC 3.2.1.3

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-Is called an Enzyme Commision Number

-Defines the function of the enzyme

-1st Number (3): Type of Reaction

-2nd Number (2): Substrate

-3rd Number (1): Catalytic Action

-4th Number (3): Specific details for the reaction

Front 28

Hydrolyzes

Back 28

GA Interacts with Starch to make Glucose

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What is Starch?

Back 29

Long polymer chain of glucose molecules

Front 30

Structure of Amylose (Starch)

Back 30

-Middle Section: Repeating Units

Front 31

What does it mean to HYDROLYZE STARCH?

Back 31

Cleave a glucose molecule from the end:

GA catalyzes the pro­duction of D-glucosefrom the non-reducing end of starch and maltooligosacchar­ides, cleaving both a-(1-4) or a-(1-6) glucosidic bonds.

Front 32

Why would we want to Hydrolyze starch?

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Glucose is an optimal substrate for a fermentation process

-EXs: biofuels, high fructose corn syrup,

1,3 propanediol, etc

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Why not just feed a fermentation glucose?

Back 33

Hydrolyzing starch is cheaper

Front 34

Where does starch come from?

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We harvest this mostly from plants (e.g. corn starch)

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Why do plants make starch in the first place?

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-Effective method of storing food reserves

-Microbes would degrade a glucose storage reserve

-Most microbes cannot degrade starch natively (some can)

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The Enzyme Assay:

Back 36

-We want to measure the amount of glucose present----> as time increases, glucose increases

-Principles of the assay (how to measure glucose?)

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Principle of Enzymatic Glucose Assay Method:

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-Glucose is phosphorylated by adenosinetriphosphate (ATP) in the reaction catalyzed by hexokinase.

- Glucose-6-phosphate (G6P) is then oxidized to 6-phosphogluconatein the presence of oxidized nicotinamide adenine dinucleotide (NAD) in areaction catalyzed by glucose-6-phosphate dehydrogenase (G6PDH).

- During this oxidation, an equimolar amount of NAD isreduced to NADH. The consequent increase in absorbance at 340 nm is directlyproportional to glucose concentration.

*NADH is easily measured using absorbance at 340 nm

Front 38

Michaelis - Menten enzyme kinetics

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Front 39

Ifthe substrate concentration is far lower than Km, [S] can be omitted in thedenominator. The equation will reduceto:

Back 39

Front 40

Solving a differential equation -d[S]/dt

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y=mx

-Plot y-axis: ln(S/S0)

-Plot x-axis: t

Plot will produce a straight line.

Slope = -k

intercept = 0

Front 41

Calculating Delta A

Back 41

Front 42

Calculating Glucose Concentration:

mg glucose/ml

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Front 43

Starch Concentration

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Front 44

What is a Protein?

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1.Polymers composed of amino acid monomers linkedtogether by peptide bonds forming a polypeptide chain.

2.They are linear polymers without branches

3.Each protein has a unique structure todifferentiate itself from all other proteins.

4.Proteins that are used as drugs are calledbiopharmaceuticals.

5.DNA codes proteins

6. DNA ->RNA -> Proteins

Front 45

What is a Polymer?

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1.A large molecule (Macromolecules) composed ofmany similar smaller molecules (monomers) linked together.

2.Responsible for the molecular"uniqueness" of an organism

Front 46

What is an Amino Acid

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Amino acids are a simple organic compoundcontaining both a carboxyl (—COOH) and an amino (—NH2) group.

Front 47

10 Amino Acids that Human can produce:

Back 47

1. Alanine(Small)

2.Asparagine (Amide)

3. AsparticAcid (Acidic)

4. Cysteine(Nucleophilic)

5. GlutamicAcid (Acidic)

6.Glutamine (Amide)

7. Glycine(Small)

8. Proline(Hydrophobic)

9. Serine(Nucleophilic)

10.Tyrosine (Aromatic)

Front 48

The ESSENTIAL AminoAcids Required in the diet:

Back 48

1. Arginine(required for young, but not for adults) (Basic)

2.Histidine (Basic)

3.Isoleucine (Hydrophobic)

4. Leucine(Hydrophobic)

5. Lysine(Basic)

6.Methionine (Hydrophobic)

7.Phenylalanine (Aromatic)

8.Threonine (Nucleophilic)

9.Tryptophan (Aromatic)

10. Valine(Hydrophobic)

Front 49

Amino Acids' Role in Proteins

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-Proteins not only catalyze all (or most) of thereactions in living cells, they control virtually all-cellular process.

-In addition, proteins contain within their aminoacid sequences the necessary information to determine how that protein willfold into a three dimensional structure, and the stability of the resultingstructure.

Front 50

Significance of Amino Acids

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- Failure to obtain enough of even 1 of the 10essential amino acids, those that we cannot make, results in degradation of thebody's proteins—muscle and so forth.

-Unlike fat and starch, the human body does notstore excess amino acids for later use—the amino acids must be in the foodevery day.

Front 51

Protein Structure

Back 51

-One or more polypeptide chains twisted into a3-D shape form a protein

-Chemical bonding between portions of thepolypeptide chain aid in holding the protein together and giving it its shape

Front 52

Protein Production

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1.Proteins need to be expressed by a biologicalhost – Expression Host

2.The process to insert a gene of interest into anexpression host is called recombinantDNA technology

3.Protein expressed by a host, which does notnaturally produce the protein, is called RecombinantProtein

Front 53

Protein Expression

Back 53

1. Many Expression hosts:

o Bacteria, such as E. coli

o Yeast

o Mammalian Cell

2. Other Expression hosts:

o Transgenic Plants: corn, potatoes, tomatoes o Transgenic Animals: Sheep, goats, chicken eggs,cows

Front 54

Protein Purification

Back 54

1. The mostcostly process for protein production

2. Typically many unitoperations are needed to purify a protein from a complex feedstock, which contains other proteins, DNAs, RNAs,and other molecules

3. The ensemble of the unitoperations is called a protein purification process

Front 55

Chromatography

Back 55

The materials inside the column is calledresins, which could have different chemical or biochemical ligands attached tothem to enable specific interactions with certain molecules – stationary phase

Proteins to be separated by chromatography arein a liquid solution – mobile phase

Front 56

Chromatogram

Back 56

A chromatogram is the visual output of the chromatography operation

Front 57

Detection of Protein

Back 57

The presence of protein in a liquid solution isoften determined by measuring the absorbance at 280 nm because all proteins(and many other compounds) absorb ultraviolet (UV) light

Front 58

Detection of Protein Visual

Back 58

Front 59

The Beer - Lambert Law

Back 59

Front 60

Protein Concentration

Back 60

* Proteinmolecular weight

-Lysozyme’sMW = 14,400 Da (Dalton)

* Alysozyme solution has a concentration of 1 mg/ml

* Calculatethe concentration in mol/L

Front 61

Mass Balances in Chromatography

Back 61

Front 62

Mass Balances in Chromatography Cont'd

Back 62

1. Determine the protein concentration in each test tube(fraction), which contains the target protein.

2. Calculate the total protein recovered:

3. Calculate the recovery (yield) of a proteinafter a separation operation:

Front 63

Gel Filtration Chromatography

Back 63

-Method to separate desired proteins from mixture

-GelFiltration – separate molecules by size

Front 64

Protein Recovered

Back 64

Front 65

Why is water such a big deal?

Back 65

Water is essential to life60-80% of the human body is waterMultiple needs: drinking, washing, agriculture, cleaning, industry, etc.

Water is a frighteningly effective disease vector

“Contaminated water can contact many more people than an infected person can…the more waterborne the diarrheal bacterium, the more deadly it is.” ~Paul Ewald, Plague Time: The New Germ Theory of Disease

Front 66

Thomas Equation

Back 66

n = number ofpositive wells for each dilution

N = totalnumber of wells for each dilution

V = volume ofa single well for each dilution

Front 67

Mass-Balance (Flow Rates)

Back 67

Front 68

Flow Rate Approach

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Front 69

Flow Rate Relative Concentration

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Front 70

Flow Rate Determining K

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Front 71

Flow Rate Sample Calculation

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Front 72

Discharge

Back 72

The volume of water flowing through a cross section of a stream channel per unit time.

*The most fundamental of hydrologic measurements that characterize all river and stream ecosystems.

Front 73

Velocity - Area Method

Back 73

Involvesmeasuring water depth and velocityat points across a stream section witha current meter.

*The most common approach to measuring discharge.

Front 74

Physical Processes that Contribute to Breakthrough Curves

Back 74

tracer mass recovered, mean residence time, mean flow velocities,longitudinal dispersion,depth, and storage

Front 75

Breakthrough Curve from Stream Tracer Lab

Back 75

The breakthrough curve displayed in Figure 2 resembled a bell-curved, showing a Gaussian distribution of the plateau concentration.

This curve demonstrates relative concentration running in the uniform course of flow.

Front 76

Stream Tracer Intro

Back 76

1. In this laboratory, wewill use a conservative solute injection technique to quantify flow rate withina stream channel.

2. A conservative(nonreactive) tracer (e.g. NaCl) will be injected into a stream channel at aknown flow rate, q­­­­i.

3. Electricalconductivity (EC) is a proxy for the salt concentration.

4. By measuring EC priorand during the injection, we can determine the stream flowrate Q.

Front 77

Whatquestion(s) was the study authors attempting to answer with this researcheffort?

Back 77

-Whatrelationships exist between the presence of enteric bacteria and demographicfactors, such as land use?

-Whateffect did different physical and chemical water quality factors have in thepresence of enteric bacteria?

Front 78

The authors state onp. 1048: “An advantage to the goals of our investigation was that the fiveseparate estuarine watersheds targeted for study were similar in climate,geography, and soil type because of inter-watershed proximity”. Why was itimportant to keep these factors relatively constant?

Back 78

-It was needed to ensure that any varyingdata was a result of what was being tested.

-Eliminating as many outside variables aspossible allows for procuring certainty so any discrepancy present in data is aresult of the testing inconstant.

Front 79

What is “turbidity”? Table 3 on p. 1050indicates that it was strongly positively correlated with fecal coliform and E. coli concentrations. Why would thisbe so?

Back 79

-Turbidity is a measure of cloudiness ofwater based on how many particles are suspended in the water.

-There would be a high correlationbetween bacteria counts and turbidity due to increase in turbidity holding agreat share in the aftermath of a storm water runoff from a surrounding human –inhabited area.

Front 80

Table 5 reveals a statisticallysignificant relationship between fecal coliform levels in streams and totalland area and human population. Where might these bacteria be coming from?

Back 80

-There are a number of fecal coliformsources present in highly populated areas including wild and domestic animalfeces, agricultural runoff from fields, and waste from sewage plants.

Front 81

5. On p. 1053 of the discussion theauthors assert: “the quality, rather than quantity, of land development is themost important influence on urban and suburban nonpoint source fecal coliformbacterial pollution.” What do the authors mean by “quality land development”?How would these affect bacteria concentrations in streams?

Back 81

-This meant that quality land development creates moreimpervious surface where water was unable to enter into the ground and leavebehind fecal matter.

-The water would instead flowmore directly into the stream and rivers, bringing with it any fecal matter itflowed over on impervious surfaces like concrete.

Front 82

Fecal Indicator Bacteriaand Anthropogenic Influences LaboratoryGOAL:

Back 82

The overall goal of this laboratory is touse observational fecal indicator bacteria data to identify five “mystery”water samples

Front 83

Five Mystery Water Samples

Back 83

·New River in Radford, VA

·Pandapas Pond in GilesCounty

·Seitz Hall bathroom sink

·Vet-Med Pond on VTCampus

·Duck Pond on VT Campus

Front 84

Colilert Defined Substrate Method

Back 84

-Colilert* simultaneously detects total coliforms and E. coli in water.

-When total coliforms metabolize Colilert’s nutrient-indicator, ONPG, the sample turns yellow.

-When E. coli metabolize Colilert’snutrient-indicator, MUG, the sample also fluoresces.

-Colilert can simultaneously detect these bacteria at 1 cfu/100 mL within 24 hourseven with as many as 2 million heterotrophic bacteria per 100 mL present.

Front 85

Sample # 4

0,0 Coliforms & 0,0 E.coli

Back 85

The sink is likely to have the lowest concentration of fecal indicators, as the water was run through human sanitation systems and cleaned extensively.

Front 86

Sample # 5

49,29 Coliforms & 7,2 E.coli

Back 86

-The duck pond, which was exclusively designed to attract ducks and other waterfowl, is likely to have one of the highest fecal indicator concentrations due to it attracting large populations of ducks.

-The duck pond was originally built as a storm water retention pond, fed by Stroubles creek. This means that all of the bacteria picked up by Stroubles creek is given to the duck pond and held there.

Front 87

Sample # 1

48,15 Coliforms & 19,2 E.coli

Back 87

-Pandapas pond is a manmade pond that is not an impaired body of water and is open for recreational use for the general public.

-Therefore it is not very likely to have large concentrations of fecal indicators.

Front 88

Sample # 2

38,9 Coliforms & 29,7 E.coli

Back 88

-According to a report released by the Virginia Department of Environmental Quality (VDEQ) sections of the New River and many runs and creeks in the New River Basin are considered impaired for E. Coli and high Mercury concentrations as of 2014.

-Since the New River runs along a very rural area, there are many farms and wild lands to allow for fecal matter to wash into its water and so it would likely have a high coliform concentration.

Front 89

Sample # 3

48,11 Coliforms & 43,2 E.coli

Back 89

Finally, the Vet-Med Pond would fall into a similar category to the duck pond, as it is a still body of water.

Front 90

Microbiology and Public Health

Back 90

Front 91

Whatare some examples of water contaminants of human health concern?

Back 91

-Arsenic

-Fluoride

-Selenium and Uranium

-Cyanide

-Lead

-Inorganic Mercury

Front 92

Prokaryotic Cells

Back 92

singlecelled organisms with no membrane-bound organelles (i.e. no ‘realnucleus’)(2-4 μm)

Front 93

Eukaryotic Cells

Back 93

cellswith a true membrane-bound nucleus (> 5 μm)

Front 94

Viruses

Back 94

Obligateintracellular parasites (20-300nm)

Front 95

Where are microorganisms essential?

Back 95

Withinour bodies

Withinnatural ecosystems

Withinengineered systems

Front 96

Pathogen

Back 96

An infectious microorganism; an etiological agent of chronic disease.

- Several distinct pathogens identified

- New diseases are emerging

Front 97

How do pathogens get into our water?

Back 97

Sanitation= provisionoffacilities and services for the safe disposal of humanurine and feces

Front 98

TheFecal-Oral Exposure Route

Back 98

-Is a route of transmission of a disease, when pathogens in fecal particles passing from one host are introduced into the oral cavity of another host.

-One main cause of fecal-oral disease transmission in developing countries is lack of adequate sanitation.

Front 99

Where are thepotential sources of fecal contamination?

Back 99

Wastewater treatment plants

On-site septic systems

Domestic and wild animal manure

Storm runoff

Front 100

Howdo we monitor microbial contamination?

Back 100

Members of two bacteria groups, coliforms and fecal streptococci, are used as indicators of possible sewage contamination because they are commonly found in human and animal feces.

Front 101

Fecal Indicator Organisms

Back 101

used in the detection andmonitoring of sewage; sentinels of

Front 102

Characteristics of an Ideal indicator Organism

Back 102

-Occurswhere pathogens do

-Canbe isolated from all types of water

-Easyto isolate and count

-Cannotgrow in the environment

-Moreresistant to disinfection than are pathogens

-Foundin higher numbers than pathogens

-Nonon-fecal sources

-Densityshould relate to human health risk

Front 103

Coliforms

Back 103

Gram-negative,non-sporulating,rod-shaped bacteria that ferment lactose; native mammalian intestinal flora

Front 104

NOTE****

Back 104

ALL Virginia surface waters are classified as

“primarycontact”

Front 105

States of the Waters

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Theleading cause of impaired surface waters in the United States is elevatedconcentrations of fecal indicator bacteria.

Front 106

Surface Water Body Impairments

Back 106

-Diseaseoutbreaks associated with drinking water have decreased

*HOWEVER,water demand is increasing * Treatmentcosts money and energy * Emergingchlorine-resistant pathogens

-Diseaseoutbreaks associated with recreational water usage have increased

-Severalrecent food-borne outbreaks associated with irrigation waters

Front 107

Process vs. Principle

Back 107

Front 108

BSE Mission Statement

Back 108

The mission of the Biological Systems Engineering Department is to develop and disseminate engineering knowledge and practices that protect natural resources and improve sustainable production, processing, and utilization of biological materials.

Front 109

BSE Design Principles

Back 109

1.Identify,quantify, and manage risk, including the risk of inaction.

2. Clearlycommunicate sources of uncertainty and inherent design limitations (includingdesign life).

3.Exercise soundleadership, ethics, management, and stewardship in decision-making processes.

4. Employ anintegrated systems approach thatincludes social, environmental, and economic considerations.

5. Promote theuse of biological materials and processesto devise sustainable solutions andprotectnatural resources.

Front 110

1)Purpose of Identifying need

Back 110

Understandingthe client’s demand

▪Need: customerwants something fun in the local park

Front 111

2) Purpose ofdefining the problem

Back 111

Develop cleardefinition of the problem

▪Question: Whatis our problem?

Front 112

3) Purpose of Search

Back 112

Learn what’sbeen done, mistakes made, possible improvements

▪Example:internet

▪Question:Where can we search?

Front 113

4) Purpose of Constraints

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Identifylimitations (physical limits, cost, performance, etc.)

▪Example:design must be delivered by July 2015

▪Question: Whatare some constraints in designing our item?

Front 114

5) Purpose of Criteria

Back 114

Identifydesirable characteristics for the design

▪Example: theitem should be durable

▪Question: Whatare some criteria that we would want for our design?

Front 115

6) Alternative Solutions

Back 115

▪Purpose:generate multiple solutions

▪Example: slide

▪Question: Whatare some alternative solutions that meet our constraints and criteria?

Front 116

7) Analysis

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▪Purpose ofanalysis: use mathematical, engineering, market principles, and common sense,to assess the solutions

▪Examples: putdesign under load

▪Swing design -What types ofanalysis can you perform?

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8) Decision

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▪Purpose ofdecision: determine the optimal solution

▪Example –Usedecision matrix to choose the swing design that meets constraints and bestmeets criteria

Front 118

9) Specification

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▪Purpose:provide details of design

▪Example:materials, industry standards required

▪Swing design:based on constraints and criteria, what specifications would you make in yourdesign?

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10) Communication

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▪Purpose:convey the problem/need, decision-making process (constraints, criteria, etc.),final decision, and details of final design

▪Examples: oralpresentation, written reports

Front 120

DESIGN LAB

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▪Objective:design a sedimentation tank that will settle kaolinite (a type of clay) quicklyand cheaply

▪In lab: smallscale sedimentation experiments

▪Design: largescale sedimentation facility ▪Dimensions oftank(s)

▪Come up withconstraints, criteria, and specifications for your sedimentation design *and*that consider the scenario provided