BMB 212 Lab Exam 2 PSU

Module 6 Part 1

Explain how recombinant DNA technology is used to produce proteins of interest

to produce large quantities of proteins like insulin

What is a recombinant DNA

circular DNA plasmid that carries essential elements that allow the DNA to function inside a cell and contain a gene of interest

Know how to draw a DNA plasmid with gene insert

Ori

replication of origin so the plasmid can replicate independent of the bacterial genome

Antibiotic Resistance Gene

-gene that codes for proteins that provide resistance to certain antibiotics

-referred to as a selective marker for bacteria to grow in plasmid containing an ARG

-ie. KanR, AmpR, etc.

Restriction Enzyme

-proteins that cut DNA at specific sequences

-recognize a DNA sequence and then cut the DNA at a site nearby

-site found inside recognition sequence

Multiple Cloning Site (MCS)

site containing a number of different RE recognition sequences clustered together

Elements to aid in gene expression

-transcription

-translation

Promoter

specific region of a gene where RNA polymerase can bind and begin transcription

Tags

-help in detection and/or purification of the proteins

-sequences found in in plasmids so that any proteins that are expressed from a sequence cloned into the plasmid can be fused to a tag

Gene coding for protein of interest

-gene cloned into the plasmid using cloning techniques

-expressed using the gene expression elements found in the plasmid

Explain the technique used to purify plasmid DNA from bacterial cells

-cut and paste cloning

-use REs to cut (digest) the empty plasmid to make a linear piece of DNA

-PCR is also used to copy the gene of interest in a test tube

Be able to analyze absorbance data to make conclusions about DNA concentration and purity

-purity:

<1.8 = DNA is contaminated

1.8-2.0 = DNA is pure

>2.0 = RNA contamination

-concentration: can be found through Beer's Law equation or conversion factor

-take conc. and multiply by total volume of isolated DNA to find the total yield

- as ABS goes up, conc. goes up

Resuspension solution contained

TE buffer and RNase

TE buffer - resuspends the cells

RNase - endonuclease which digests the RNA and facilitates its separation from DNA

Lysis solution used

NaOH/SDS

SDS - denatures bacterial proteins

NaOH - denatures chromosomal and plasmid DNA

Neutralization solution contained

K acetate and salt

to precipitate protein

Neutralizes cell extract

salt - helps bind to membrane

Wash buffer used

ethyl alcohol

removes weakly bound molecules with positive charges

keeps plasmid DNA bound to the spin filter and washes away impurities

Elution buffer contained

10mM Tris

ABS of nucleic acids

260nm

ABS of proteins

280nm

Module 6 Part 2

Describe a restriction digest

cleaves the plasmid DNA at specific recognition sites

Describe how a restriction digest can be used to determine identity of a mini prepped plasmid

can analyze the DNA fragments to find differences in the DNA

ex: one recognition site = one fragment

How is DNA visualized in a gel

-use fluorescent dye

-we used EtBr (ethidium bromide)

How does agarose percentage relates to DNA separation

a lower agarose percentage gives better resolution and separation of higher molecular-weight bands

How do DNA fragments separate on a gel based on shape and size and how does this relate to plasmid DNA

-larger molecules move through the pores that form when the gel solidifies slower than smaller molecules

-nicked circles = single break in a phosphodiester bond; run slowest; can make an open circle structure (hula hoop)

-linear = linear form of supercoiled (stretched out)

-supercoiled = circular plasmids purified from bacterial plasmid in a high-quality state; run fastest (rolled tight in a ball)

Know how to predict fragment sizes from plasmid maps

Size of the fragment is the change in size of the plasmid with the fragment added

Know how to interpret data from restriction digest on an agarose gel

-know which one contains the empty plasmid or the gene of interest plasmid. Empty plasmid will not contain a 1 kB band

Module 7 Part 1

Describe the steps in gene expression

-transcription: DNA to RNA; RNA Polymerase assembles at promoter sequences along w/ trans. factors and then moves forward until it finds a trans. start site where it then reads the DNA and uses code to make a specific RNA molecule

-translation: RNA to protein; If the RNA Polymerase binds and reads the T7 promoter sequences in the pET30:GFP plasmid, the RNA polymerase will read the GFP gene and make the mRNA for GFP. Then other bacterial proteins will read the GFP mRNA and make the GFP protein --> fluoresce green

Describe how promoter sequences play a role in the expression process

-promoters "signal" polymerase to express gene (binds to polymerase)

-controls expression

Describe how E.coli cells are genetically engineered to allow for expression from the T7 promoter

Need to place plasmid into a specific strain of bacteria that have previously been genetically modified to express the T7 RNA Polymerase

Why is genetic engineering of the E.coli necessary to get expression of genes that use T7 promoter

The T7 promoter is not the natural E.coli promoter, which means that RNA Polymerase cannot read and bind to the T7 promoter

Explain how the lac operon sequences (lac promoter, operator, and lacl gene) are used to control gene expression

-lac promoter: inducible; "turned on" or "turned off" based on the presence of certain chemicals

-lac operon: cluster of genes found in E.coli; produces the enzyme lactase to break down lactose

-lacl repressor: binds to DNA sequence (lac operator) to act as a roadblock; signals promoter to ignore the sequence causing RNA polymerase to be blocked because it's not being controlled to express gene, so the promoter moves to the next available sequence to code (operator) and prevents transcription; can move to transcription start site for the lactase gene

-when repressor no longer ignores signal to express gene, RNA Poly. continues down the chain to transcribe/code by polymerase

Describe the role of IPTG in gene expression

-lactose analog

-looks like lactose molecule but cannot be broken down

-will continuously induce GFP production, leading to the higher yield (unlimited)

-IPTG binds to the lac operon repressor (which inhibits the expression of the gene encoded in lac O) thus releases the inhibition, leading to the production of T7 RNA Polymerase

Be able to make predictions about when genes would be expressed to significant levels if they are controlled by sequences such as T7 promoters and lac operators

-lac O can be inserted to control expression of the T7 polymerase

-no lactose around, the lac repressor protein will sit on the lac operator DNA sequence and block the ability of the T7 RNA polymerase to express the gene found after that T7 promoter and lac operator sequence

Module 7 part 2

Describe steps necessary to purify a protein from an organism like bacteria using an affinity purification approach

1. make protein extract before purifying

2. bind protein of interest to an affinity matrix

3. wash away any unbound/loose contaminating proteins

4. elute to release protein from the affinity matrix

Centrifugation

-separates lysate from insoluble cell debris

Lysis

-break down

-used BugBuster to release soluble proteins from bacteria, yeast, etc. cells

His-GFP Purification

-only performed on induced cell lysate (higher levels of GFP)

-bind, wash, elute

Explain the use of a his tag in affinity purification

-his tags have a high affinity towards Nickel ions, so they bind together

-his tags (GFP) are eluted during the elution step with the buffer containing histidine's analogue, imidazole (used in high concentration)

Describe primary structure of protein and how it allows for GFP fluorescence

-sequence of a chain of amino acids

-adopts a beta sheet rich secondary structure

Describe tertiary structure of protein and how it allows for GFP fluorescence

-3D folding pattern of a protein due to side chain interactions

-goes through a series of chemical reactions to form the internal chromophore that allows the GFP protein to fluoresce under UV light

Describe secondary structure of protein and how it allows for GFP fluorescence

-local folding of the polypeptide chain into helices or sheets

-final arrangement of the beta sheets results in tertiary structure

Draw a picture that relates part 2 to part 1 of module 7

Module 7 Part 3

Explain the theory behind SDS-PAGE and how proteins are separated

-allows separation of proteins by size through a polyacrylamide matrix

-pore size is major difference between SDS-PAGE and gel electrophoresis

-separated by molecular weight NOT by shape due to addition of a detergent (SDS) which denatures the proteins prior to and during electrophoresis run

-denaturization causes PRIMARY structure to allow the DNA molecules to flow easily

Describe the purpose of the reagents used in SDS-PAGE as well as the visualization process

4X Protein Sample Buffer

-2-mercaptoethanol is another reducing agent that denatures the protein by breaking disulfide bonds

-SDS also denatures the protein by having a head group giving the protein a negative charge (anionic detergent)

12% Mini-PROTEAN TGX poly-acrylamide gels

-allows for separation of polypeptides (proteins)

-contains a crosslinker that creates the 3D lattice

-12% = separate at ~12-200 kD range of molecular weights

Precision Plus Protein Kaleidoscope

Prestained Protein Standards

-contain multi-colored proteins to visualize separation and provide a size reference

SDS Running Buffer

25mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3

-Tris = buffering reagent

-Glycine = AA with a charged state for separation (critical)

-0.1% SDS = keeps proteins coated to maintain uniform charge of proteins

Coomassie Stain

-visualizes proteins on the gel

-contains methanol and acetic acid to fix (keep) the proteins in the gel during staining/destaining

Destain

-contains methanol and acetic acid which causes Coomassie dye not attached to proteins to come out of the gel

Smaller protein =

higher gel %

Use protein standards to estimate size of protein on an SDS-PAGE gel

know the ladder found in manual

Analyze data and make conclusions about the effectiveness of a purification scheme

-If gel done correctly, will see different bands appear

-If E1 has more than 1 band, then protein was not pure

-W1, W2, and W3 should all have no bands or very faint bands since the contaminated proteins are washed off

Explain the relationship of the SDS-PAGE portion of this module with the previous module on DNA and with the previous parts of the protein module

SDS is used for purifying and analyzing proteins; previous electrophoresis shows relative sizes of any molecule and shows if the gel plasmid contained the gene of interest

Explain how to access information on protein sequence and analyze that sequence for molecular weight

use online external tool

Module 8: Lipids

Be able to describe the chemical reaction that occurs during saponification

-saponification = the alkaline hydrolysis of a triacylglycerol

-NaOH is used to cleave the fatty acid ester linkages

-evaporate the solvent (ethanol)

Predict how the use of different types of oils will result in "harder" or "softer" soap based on the chemical properties of the lipids in that cell

-harder soaps = saturated FAs; more interactions

-softer soaps = polyunsaturated FAs

Importance of fatty acids

-energy source

-temperature

-cell membrane structure

Fatty acids with higher melting points are removed...

leaving the fatty acids with lower melting points