MCB4034L Final Review

What is the purpose of sequence analysis in this experiment?

To determine if there are mutations in the sequence encoding the GFP-CBM fusion protein

What region of the plasmid is flanked by the T7 and T7-term primers?

The region of interest in plasmid pET28a

What types of files are received per plasmid in Sanger sequencing/

- Ab1 files: Chromatograms (raw data) for T7 and T7-term sequences

- Seq files: Text format extracted from ab1 files for both primers

What are the two data files of interest in ONT sequencing?

- FASTA files: contain the actual sequence

- HTML files: show the plasmid map

How does ONT sequencing differ from Sanger sequencing?

It is more straightforward (ex: no reverse complements), but you have to locate the sequence of interest within the full plasmid

Expression of the GFP-CBM fusion protein General Steps

1. Select a plasmid w/ a correct sequence + adequate residual plasmid after sequencing

2. Transformation of competent cells

3. One transformed colony selected fro overnight cultures in liquid LB Kan30 Cm20 medium

3. 2mL is used to inoculate 400mL culture + incubated until OD600 between 0.6 + 1.0

4. Sample collected for polyacrylamide gel electrophoresis

5. Induction of gene expression with the addition of IPTG to a final concentration of 1mM

6. Continue bacterial cell culture for 24-48 hours at 30C

What type of cells are transformed?

E. coli BL21 (DE3) pLysS competent cells

How is kanamycin resistance expressed?

Through a 1- hour incubation at 37C after transformation

What medium is used for overnight cultures?

LB Kan30 Cm20 (Kanamycin + chloramphenicol)

What is the purpose of IPTG addition and at what concentration?

To induce gene expression at 1mM concentration

How long is the culture incubated after IPTG induction?

24-48 hours at 30C

- During this time the E. coli cells produce the recombinant protein

What is IPTG chemically?

Isopropyl-beta-1-D-thiogalactopyranoside

How does IPTG work as an inducer?

It mimics allolactose and binds to the lacl repressor, preventing it from binding to the lac operator

Why is IPTG a continuous inducer?

It cannot be metabolized by cells

What two roles does IPTG play in expression systems using E. coli BL212 (DE3)?

1. Induces expression of T7 RNA polymerase (under lac promoter in chromosome)

2. Activates expression of GFP-CBM cDNA via T7 promoter in plasmid

What is the first level of control in recombinant protein production?

T7 RNA polymerase under the lac promoter (needs IPTG for transcription/translation)

What is the second level of control in recombinant protein production?

The recombinant protein sequence under the T7 lac promoter (also needs IPTG)

Why is T7 lysozyme important?

It inactivates low levels off T7 RNA polymerase, preventing leaking expression

What plasmid encodes T7 lysozyme?

pLysS or pLysE

What is the difference between pLysS and pLysE?

- pLysE: Higher T7 lysozyme = more control, but slower cell growth

- pLysS: Lower T7 lysozyme = less control, better growth (maintained w/ chloramphenicol selection)

Codon bias

An organism's preference for specific codons encoding the same amino acid, reflecting tRNA abundance

Why might eukaryotic genes not translate efficiently in E. coli?

They may use codons that correspond to rare E. coli tRNAs

What do Rosetta cells contain to solve this issue of eukaryotic genes not translating efficiently in E. coli?

A plasmid encoding tRNAs that are common in eukaryotes

What is the purpose of mixing the 30mL induced culture by inversion?

To ensure a homogenous suspension of cells

After mixing, what should be done with a portion of the induced culture?

Set aside and store on ice

What is done with the remaining cells?

Harvested by centrifugation; the cell pellet is kept and the supernatant is discarded (harvested cell)

What is the ratio of lysate to cell paste used?

5 mL of lysate per gram of cell paste

Preparation of a cleared lysate

1. Add 5mL per gram of cell paste

2. Gently pipette up and down to re-suspend the pellet

3. Spin for 15 min to pellet cell debris

What is chromatography used for?

Separation of individual compounds/proteins from complex mixtures

What are the 4 main types of protein chromatography?

1. Affinity

2. Gel filtration (size exclusion)

3. Hydrophobic interaction

4. Ion exchange

What are the key components used in chromatography?

A resin (polymer beads) and a buffer that may change during purification

What is attached to the solid beads in affinity chromatography?

A specific ligand (antibodies, metal ions, enzyme substrates, the beads themselves) that interacts with the target protein

What ligand-protein interaction is used in this experiment?

His-tag of the protein binding to nickel ions

General Procedure of Affinity Chromatography

1. Mix protein extract w/ chromatography resin (binding step)

2. Load resin onto a column

3. Collect + discard flow-through

4. Wash resin to remove loosely bound proteins

5. Elute target protein using buffer that disrupts binding

6. Collect eluate in fractions

Why are eluates collected in fractions

To increase the protein concentration in each collected sample

What is the separation principle in ion exchange chromatography?

Proteins are separated based on their net charge

What determines a protein's net charge?

The side chains of amino acids, especially those exposed on the surface

Which proteins move faster through the column in ion exchange chromatography?

Neutral proteins or those with the same charge as the resin

What is the separation principle of hydrophobic interaction chromatography?

Proteins are separated based on surface hydrophobicity (hydrophobic = binds on resin beads, hydrophilic = do not bind and elute)

What is the principle of separation in gel filtration chromatography?

Proteins are separated by size

- Small = move slowly entering bead pores

- Large = move fast, bypassing pores

Opposite of gel electrophoresis

What material is used for IMAC?

Agarose beads decorated w/ nickel ions

How does the His-tagged protein interact with the column

The His-tag binds specifically to nickel ions on the beads

What happens after the protein binds?

Other proteins are eluted using a buffer with imidazole

What does SDS-PAGE stand for?

Sodium dodecyl sulfate - polyacrylamide gel electrophoresis

What does SDS do to proteins?

Adds a uniform negative charge so proteins separate purely by size

How does PAGE differ from agarose gels in composition?

- Polyacrylamide: 7.5-12% (thin, vertical)

- Agarose: 0.7-4% (thicker, horizontal)

PAGE vs. Agarose for buffers + voltages

- PAGE: Tris-Glycine-SDS; runs @ 200-300V

- Agarose: TAE/TBE/SB; runs @ 60-100V

PAGE vs. Agarose for a typical run

Both for 30-60 minutes

Can polyacrylamide gels be used for DNA?

Yes, without SDS and with a different buffer (offers higher resolution than agarose)

- Agarose used exclusively for DNA

Three staining methods for proteins in gels?

- Coomassie Brilliant Blue

- Silver staining

- Proprietary methods (which we used)

Why is a size marker used?

To estimate protein size by comparison to known molecular weights

- Usually "pre-stained" and multicolored

Coomassie Brilliant Blue Pros/Cons

Pros: Cheap, easy

Cons: Smelly, tedious (medium sensitivity)

Silver Staining Pros/Cons

Pros: High sensitivity

Cons: Toxic waste (staining procedure)

How does the BioRad Gel Doc EZ Imager visualize proteins?

UV light excites a reagent that reacts with tryptophan residues, producing fluorescence

Advantages of BioRad Gel Doc EZ Imager

Quick, no staining/destaining required, produces a digital fluorescent image

What is the main purpose of SDS-PAGE in protein analysis?

To separate proteins based on size + monitor the complexity of samples, determining the success of purification processes like chromatography

What are the four protein samples compared in this experiment?

1. Un-induced culture: Has soluble + insoluble proteins before induction (no GFP-CBM produced)

2. Induced Culture: Has soluble + insoluble proteins after GFP-CBM induction

3. Crude Lysate: Soluble proteins sued for IMAC purification

4. Eluted Sample: Ideally contains only purified GFP-CBM protein

SDS-PAGE Sample Preparation

1. Add 4x sample loading buffer

2. Heat 5 min @ 95C in waterbath

3. Transfer to ice

4. Quick centrifugation spin to collect condensation

5. Keep samples on ice + load gel + apply a constant voltage of 200V for ~40 min

Why are proteins transferred to a nitrocellulose membrane after SDS-PAGE?

B/c the gel is fragile and mostly water

What is western blotting used for?

Detection of specific proteins on a membrane using antibodies

Difference between western, southern, and northern blots?

- Western: Detects proteins (via antibodies)

- Southern: Detects DNA

- Northern: Detects RNA

Why is protein identity not always reliable by size alone?

B/c different proteins can have similar molecular weights, and low-abundance proteins may not be visible on gels

What is a Turbo Blot?

A fast, pre-packaged transfer system that moves proteins from the gel to the membrane in minutes instead of hours

Why is the Turbo blot preferred over traditional methods?

It's a faster, more convenient, and uses prepared membranes + buffers

What is the role of the primary anitbody

It binds specifically to the target antigen (protein of interest

What is the role of the secondary antibody?

It binds to the primary antibody + is enzyme-conjugated for signal detection

Why is secondary detection considered more sensitive?

Multiple secondary antibodies can bind to one primary, amplifying the signal

Why is primary-only detection sometimes preferred?

It's faster + more direct, but less sensitive

What is the main difference between colorimetric + chemiluminescent detection?

- Colorimetric: Produces a visible color on the membrane

- Chemiluminescent: Produces light detected on film/screen

Which detection method is used in this lab?

Colorimetric detection

Colorimetric Detection Steps

1. Primary antibody binds to the target protein (antigen)

2. Enzyme-linked secondary antibody binds to the primary

3. The enzyme converts the substrate into a colored precipitate visible on the membrane

- A purple precipitate forms where the enzyme-antibody complex is located

Chemiluminescent Detection Steps

1. Secondary antibody w/ alkaline phosphatase (AP) binds to primary antibody

2. AP converts a chemiluminescent substrate, producing light

3. Light exposes an X-ray film or phosphor screen

- emission of light from the enzyme reaction is recorded as black bands

Which phrasing correctly describes a bacterial transformation?

- "I transformed E. coli w/ the plasmid encodign GFP-CBM"

- "I introduced the plasmid encodign GFP-CBM into E/ coli"

Which phrasing is incorrect?

" I translated the plasmid encoding GFP-CBM into E. coli"

What antibody was used for detection?

HRP-conjugated mouse anti-6xHis tag antibody

What are the main steps in imumunodetection after transfer?

1. Block membrane in blockign solution

2. Incubate w/ antibody (1:1,000) for one hour

3. Wash 3x10 min in PBS + 0.05% Tween-20

4. Rinse 5 min in PBS (no detergent)

5. Perform colorimetric detection

Why is PBS used for washing + blocking?

It mimics blood serum conditions, maintaining protein stability

How are monoclonal antibodies produced?

1. Inject mouse w/ the antigen of interest

2. B-lymphocytes in the spleen produce antibodies

3. B-cells are isolated + fused w/ myeloma (cancer) cells

4. Hybridoma cells that produce the desired antibody are selected + cultured

What is a hybridoma?

A fusion between a B-cell and a myeloma cell that produces monoclonal antibodies indefinitely

What are the steps in colorimetric detection on a membrane?

1. Add detection substrate

2. Monitor color development

3. Rinse in water to stop the reaction once sufficient color forms

What are the two functional domains of the GFP-CBM fusion protein?

- GFP domain: Provides fluorescence (visual confirmation)

- CBM domain: Enables binding to cellulose

What does the functional assay verify?

That both domains (fluorescent + binding) of the recombinant protein are functional