Lab: Genetics 2 - Gel Electrophoresis and Gene Regulation

Gel electrophoresis 

• a method used to separate macromolecules like DNA, RNA, and proteins 

• used for visualizing and identifying DNA fragments

How do fragments in gel electrophoresis separate?

based on size, shape, and charge

What charge will DNA move towards in an agarose gel electrophoresis?

when placed in an electrical field, DNA will migrate towards the positive pole (anode)

How does size affect separation of DNA fragments in gel electrophoresis?

smaller fragments move faster than larger ones

How does shape affect separation of DNA fragments in gel electrophoresis?

supercoiled fragments take longer compared to linear DNA fragments

How does charge separation of DNA fragments in gel electrophoresis?

DNA mores away from negatively charges cathode and towards positively charged anode

What do we use in lab to stain nucleic acids in electrophoresis gels?

SafeGlow

DNA ladders

used to compare the size of your DNA band to ladder with known sizes of DNA, allowing the identification of sample fragments

What do you use to identify an unknown DNA segment from agarose gel electrophoresis?

a semi-log graph - one axis is exponential 

gene regulation

how each cell type can express different genes despite having all 46 chromosomes, cells express only a subset of genes

What are the components of the lac operon?

1. promoter

2. cap and operator 

3. lacl 

4. lacZ

5. lacY

6. lacA

promoter

where RNA polymerase binds to to transcribe the genes that follow the promoter

CAP and operator

molecules will bind to repress or enhance the expression of the downstream genes

lacl

codes for the lac repressors, comes way before the promoter

lacZ

encodes for the enzyme B-galactosidase which hydrolyzes lactose, and converts lactose in allolactose 

lacY

encodes for permease which transports lactose into the cell

lacA

functions as transacetylase which removes excess sugar from the cell

What happens when there is no glucose?

cAMP combines with CAP to create the CAP-cAMP complex which enhances the transcription of the lac operon genes

What happens if glucose is present?

cAMP levels are low, so little cAMP-CAP complexes are made, and transcription of the lac operon will still happen but at very low levels

What happens if no lactose is present?

no allolactose is produced and the lac repressors is not inactivated, so it binds to the operator and stops transcription of the lac operon 

What happens is lactose is present?

allolactose will inactivate the lac repressor and transcription of the lac operon will occur

What is the positive regulation of the lac operon?

the cAMP levels as a result of glucose levels

What is the negative regulation of the lac operon?

the presence of lactose

Why is just negative or positive regulation not sufficient?

negative: without the presence of lactose, the lac repressor cannot be inactived and transcription cannot occur

positive: without high cAMP levels, the transcription of the lac operon genes will not be efficient