Lecture 10-11 Tools for analyzing gene expression (copy)

Model Organisms

Features

• cheap and common

• easy to propagate

• easy to manipulate

Bacteriophage lambda

The bacterium Escherichia coli

Transient and stable transfection assays

• plasmid DNA remains extrachromosomal or integrated

• Transient transfection

  • intro of DNA into cells for little time

• Stable transfection

  • cells selected for stable integration of plasmid into chromosome by drug resistance

Transient transfection

• common

• not stable

• less predictable

• less long-term seconday effects

Stable transfection

Drug resistance mRNA

Reporter Genes

Def

• known gene whos RNA or protein level can be measure easily/accurately

• used to replace other codign regions whs protein products are difficult to measure quantittively

Application

• study regulatory elements of genes

• effeciencey of gene delivery system

• GFP- intracellular fate of gene product

• FRET- Protein-Protein interactions

• GFP- DNA0protein interactions

Purification and detection protein using epxression tags

• reporter gene attached t other sequence so that reporter protein is synthesized and fused to another person

• often shrt peptide sequence that acks as affinity or epitope tag (antigenic determinant)

• Commonly used affinity tag:

  • Histidine (His) tag: 6-histidine

  • GST tag: gluthathione-S-transferase

  • MBP tag: maltose-binding protein

• Application:

  • Protein purifiicaion

  • Protein localization

  • FRET- Protein-protein interactions

His tags

• small tag

• easy to add

• not interfere with protein functions

GST tag

• can interfere with protein function

• can remove enzymatically

MBP (maltose binding protein)

• increase protein solubility

• interfere with protein function

• removed enzymatically

Immunotags

• c-Myc

• FLAG

• HA

Fluorescent protein tags

• Fluorescence of GFP detected in vivo and can be expresed everywhere

• application

  • localization of protein of interest

  • reporter assay to study a promoter

  • FRET- to quantify calcium

• gene cloned by Douglas Prasher

Flourescence terminology

• Fluorochrome: dye or molecule that exhibits fluorescence

• Fluorophore: group of atoms that absord light energy and produce color

Florescent protein with different spectra

1. Mutated GFP

   • enhanced GFP (EGFP)

2. Red flourescent protein

   • DsRed

Confocal microscpy

• ilumination achieved by scanning one or + beams of light from laser across specimin

• Collect Z series to create high resolution 3D images of sample

In Vitro mutagenesis

• Site-directed mutagenesis

  • intro of specific base sub/insertion of defined sited in a cloned DNA molecules

• Non-site directed mutagenesis

  • random mutagenesis

Analysis at level of gene transcription: RNA expression and localization

• Constitutive expression: the gene is expressed at all times.

• Spatial expression: the gene is only expressed in specific tissues in an organism.

• Temporal expression: the gene is only expressed during a specific time in development.

Technique to monitor mRNA levels

• Northern blot

  • one gene per probe

  • detects RNA,DNA, Protein with Ab

• in situ hybridization

  • localization

  • abundance

• Reverse transcription- PCR

  • first strand cDNA reandom primer or poly (dT)

• Quantitative real-time PCR (Q-PCR)

  • Ct reflets mRNA abundance

• Next gen sequenceing (systematic)

qpcr (quantitative real-Time PCR

4 steps

1. denature to create single strand

2. annel

3. tention to create product

4. use Syber green dye to annel with doubel strand DNA to emit light that is detected by machine to check for product

a sample run 10 cycles to create 10,000 molecules (faster bc less amount of cycles)

b sample run 12 cycles to create 10,000 molecules

cycle^(-small cycle-long cycle) => faster and

• 2^(-(10-12))

curve: product is s

Analysis at the level of translation: protein expression and localization

Protein gel electrophoresis

• Polyacrylamide used to make gel for better resolution rather than agarose

• AA in proteins not all charged

• Negative charge provided by binding socidum dodecyl sulfate (SDS) during sample preparation and running

• amount of SDS bound proportional to AA #

  • One-dimentional (1D) SDS-PAGE

    • separated proteins by size

  • 2D PAGE

    • sep proteins by charge and size

  • Native vs denaturing

Technique for monitoring protein levels

• western blot

  • detects protein via Ab

• In situ analysis

  • indirect immunoflourescene assay

    • single cell level

    • using Ab to detect protein

• ELISA (enzyme-linked immunosorbent assay) (liquid western blot)

  • not single cell

  • use Ab to bind to antigen and another Ab to detect signal

  • uses 96 well plate

  • enzyme generates light that is proportiona to the protein binding t antibodies

• Constructing fusion proteins with easy-to-detect tag

Antibody

• Polyclonal Ab:

  • antibody made of B cels and recognie multiple pitopes

• Monoclonal Ab: made of clonal amp of single B cell and recognise 1 epitope

Analysis of DNA- protein interaction

• EMSAL Electrophoretic mobilit shift assay

  • native gel

  • look at interaction to look at what gets bigger using native gel

• DNase 1(Deoxyribonuclease 1) footprinting (footprint is where the part that wasnt degraded when adding the Dnase is attached to proteins)

  • special for DNA-Protein interactions

  • get negative DNa then label 1 strand, then DNase 1 added to cut at random to create a band

  • end labeling

• Chromatin immunoprecipitation (ChIP) assay

  • protein-dna interaction

  • use ab to pull down protien specifically chromatin

Analysis of protein-protein interaction

• pull down assay

• yeast two-hybrid assay(in exam)

• Coimmunoprecipitation assay (CoIP)

  • Western

    • pull down protien x and then use a labeled

  • Mass spectrometry

  • pull down spike protein and sequence it

• FRET (Fluorescence resonance energy transfer)

yeast 2-hybrit screening

• GAL4 is transcription factor

     Interaction Present (ON state):

      •    Protein X (bound to GAL4 DNA-binding domain) and Protein Y (bound to GAL4 activation domain) interact.

      •    This interaction brings the activation domain close to the DNA-binding domain, enabling the recruitment of RNA polymerase and transcription factors.

      •    Result: The lacZ gene is expressed, and a color change (e.g., BLUE) is observed.

      2.    No Interaction (OFF state):

      •    Protein X and Protein Y do not interact.

      •    The GAL4 DNA-binding domain and activation domain remain separated, preventing transcription of the lacZ gene.

      •    Result: No reporter gene expression (e.g., WHITE colony).

FRET

You emit at only wavelength but express a highter wavelenth due to the first protein binding with other protein that leads to transfer of energey and emits at higher template