BMSC 240 Midterm and Final

is the study of the world using scierntific method. is the study of living things.

variables are what are altered in the experiment, while variables are what you are observing in the experiment. Both of these are known as variables.

A pH meter must be rinsed and stored in between uses. The random errors that can come from using a pH meter are not , and measuring before . The systematic errors that can come from using a pH meter is if it is not correctly.

An is a proton donor, while a is a proton acceptor.

At a low pH, both ends of the amino acid are . At high pH, both ends of the amino acid are . At moderate pH, the net charge is .

: A tube that holds the sample
Transmittance: the proportion of light that the sample allows to : the amount of light the sample blocks
: a comparison sample with non of the measure substance
Standard Curve: A series of samples used to generate comparison data

The Bradford assay uses dye. Unbound dye has an absorbance max of . Bound dye has an absorbance max of . The upper limit for linear detection is 1 mg/mL.

Gram positive cell walls are up to thick, while gram negative cell walls are only thick. Although gram negative have smaller layers, they contain a second . Gram positive cell walls also contain to keep layers together and increase the charge of the cell wall.

A is a charged molecule that interacts and makes it hard to wash off.

The steps of a gram stain are:
1.
2.
3.
4.
The mordant in a gram stain is .

A is neatly organized while a is unorganized or loose. These are both known as . To stain, a charged dye, such as , is required. The cells are not fixed to slide and the dye creates a for stain to get everywhere except on the . This requires staining to visualize , such as .

A is one where all chemicals are known and quantified, while a is one where we are not aware of every chemical.

MacConnkey Agar selects against gram using bile salts and crystal violet. It contains and . Fermenters turn on the plate. MSA has a and contains mannitol. Fermenters turn on the plate. It is useful for detecting . BAP contains 5-10% and tests for activity. The may be enhanced in environments. contains a high amount of glucose (2%). It selects for yeasts and . SIM Deep's are used to differentiate cells from , and . An test is used to see if the organism can use oxygen to generate . A result is blue/purple.

and are cell wall inhibitors. They contain a beta that mimics the substrate to link peptidoglycan. This causes the cell wall to be weaker. Natural penicillin works only against gram cells.

include: rifamycin or quinolones. Rifamycin inhibit and are highly to the cell. Quinolones inhibit which inhibits DNA replication.

An is a protein that acts as a catalyst for a chemical reaction. The makes the reaction more by lowering the .

is how fast substrate is converted into product. Over time, reactions will slow down as decreases increases and are lost to . The is used to get non-attenuated rates.

Km is the concentration of that leads to half-maximal .

: breaking the cell apart with sound waves that have high amplitude and frequency. May damage the sample as it will the sample.
: increasing pressure from a piston and releasing to break cells apart
: Putting into a hypotonic solution to put the cell in shock and causing them to burst. Good for cells who do not have a .
: Using an enzyme to damage cell walls, but is not broken apart.
: Amphipathic molecules that break down fats and oils by forming micelles
: Using force to break things apart (blender)

The advantages to using controls in an experiment are that the results can be verified and experimental procedures can be . An experiment should contain both and controls to ensure it is working. Some additional concerns that a scientist may have are:
1. Are the experiments ?
2. Are there adequate notes of ?
3. Is the experiment ?
4.Is a test or ?
5. Is a result t?
6. Can you rule out correlation from causation? Correlation is the mutual of two or more things, parts, etc.:
Example: Studies find a positive correlation between severity of illness and nutritional status of the patients.

A pH meter uses the produced by the to compare with and reports a . If the glass probe is , there is no longer a need to travel the and the will change. A scale is primarily used to chemicals in the lab. A stir plate has coated place in solutions.

The basic microscope can visualize objects down to micrometers. The microscope consists of , , , , , and .

DNA absorbs UV light at . Absorbance of UV light for proteins depends on their , but is approximately . The 260/280 ratio for pure protein is .

The chemical needs of a cell are:
: the backbone of most cells
& Hydrogen
Nitrogen: All acids
Phosphorus: , ,
Sulfur: , and
Calcium: , and
Trace elements: Enzymatic

Purification by properties:
Solubility:
Ionic Charge: chromatography, electrophoresis, isoelectric focusing
: Reverse phase and hydrophobic chromatography
Size: Dialysis, -exclusion chromatography, gel
: affinity chromatography

In an electrophoresis, velocity is directly proportional to and inversely proportional to and .

SDS-PAGE stands for gel electrophoresis. SDS is a detergent that will proteins and give them a charge. Acrylamide is a plastic monomer, used industrially that can be chained together. Acrylamide is found to be and is approximately micrograms/g in one bag of chips. Toxic effects are seen at mg/kg. You would require bags of chips in one day to die if you weighed 75 kg.

Protein samples are boiled for minutes in 5x loading buffer to be denatured. They are then coated with 10% , 20% , 0.2 M Tris-HCl pH 6.8, 0.05% blue, 10 mM B-mercaptoethanol. Gel is cast by mixing acrylamide, bisacrylamide, APS, TEMED, SDS and buffer. It is poured between glass or plastic plates. will inhibit the polymerization.

A subset of lab IgG Uses:
1. : To determine quanitity and bad identity on a gel
2. : To determine how much of a protein is present in a mixture
3. : to determine the identity of a pathogen
4. : To determine antigens/antibodies present in a sample
5. staining: To localize proteins in a cell

Plasmid Types
plasmids- transmitted during conjugation, carry a variety of information
plasmids- protect against environmental factors, multiple drug resistance plasmids
plasmids- codes for proteins that kill other microbes
plasmids- contain genes for novel catholic enzymes
- increases the pathogenicity of a bacteria

Typical components of natural plasmids are:
1. of to allow for more production
2. selection gene to force cells to keep the plasmid at high numbers
3. gene to allow differentiation from normal cells compared to plasmid containing cells
4. sites to allow places to insert foreign DNA

Assays should be:
Sensitive

Rapid

The following are important volume concepts in chromatography:
Vt- volume of the
Vo- volume the beads
Vi-volume the beads
Ve- volume at which the sample is

A gel is used to down movement, prevent of the proteins and create

Acid urea gel contain running buffer contains 5% . It will protonate some R-groups, giving many proteins an overall charge. It contains a high content of 15%. It runs slower that an SDS-PAGE and .

Native gels mobility of proteins depends on , and . The charge depends on the buffer used. The gel runs substantially than SDS-PAGE and some sample may not enter the gel based on charge at their specific pH.

is a method to look for a protein of interest from a gel using antibodies against the protein. A protein gel i first run. The gel is then transferred to a membrane with a high for protein such as transfer or transfer. The membrane has a high affinity for protein so all empty spots much be filled with or Powder. Once blocked, the antibody may be added to blot and allowed to bind A protein may then be used to detect the presence of the primary antibody. They are helpful for detection of a from a sample, profiles of protein, verification of a organism, or to detect infections, such as .

Fluorescence microscopy:
Culture cells or tissue sections are placed on .
Cells are fixed using or methanol/acetone and rinsed.
Cells are permeabilized using dilute .
Cells are blocked to reduce binding.
antibody is added/washed
fluorescent antibody is added/ washed.

Types of Fluorescent Microscopy:
Standard: whole image is illuminated and out of focus light makes the in focus image
Pro:
Cons: and
Confocal: whole image is illuminated and out of focus light is
Pro: very good
Con:
Two photon: less energetic light is used (infrared) and photons must hit the dye simultaneously to it
Pro: lower photo damage, better tissue
Con: , somewhat resolution to confocal

Typical components of vectors:
Original of
selection gene
Insert gene
sites

A concentration allows for larger band to be resolved while small bands run through. A concentration allows small bands to be resolved while large bands stack together. occurs as large DNA (>25,000) reaches the resolvable limit and otherwise will not . The DNA will travel way like a snake and no longer . Thus, the edge determine the speed and cannot change.

To isolate the plasmid:
1. Culture pelleted to remove broth
2. Cells are resuspended in , EDTA, or Salt
3. Cells are lysed with things such as NaOH, , Lysozymes,
4. Solution is treated to debris
5. Spin out debris, remains in supernatant

Plasmid precipitation:
1. Salt added to supernatant-, LiCl, NaAc
2. 1-3 volumes of alcohol added to solution- or
3. Chill on ice
4. Spin in centrifuge to collect DNA. The DNA is now found as the .
5. Wash with 70% (to desalt)
6. Dry and resuspend in water or TE. Typically a buffer is chosen to provide .