CHEM154 Midterm

Which amino acids in a protein allows it to absorb light at 280 nm?

the presence of tyrosine and tryptophan residues

In what direction do polymerases read vs extend?

-they extend in the 5' -> 3' direction but read in the 3' -> 5' direction

What are the two strands of DNA in transcription and which strand gets read by the RNA polymerase?

-there is the coding strand and there is the template strand
-the template strand gets read by the RNA polymerase

Is the mRNA complementary (with the exception of 'U's instead of 'T's) to the coding or template strand?

template

What serves as the recognition site by the RNA polymerase in transcription?

the promoter

What is the start codon and amino acid?

AUG (methionine)

What is at the 5' and 3' ends of the mRNA?

-at the 5' end there is a methyl cap
-at the 3' end there is a poly-A tail

What represents the N-terminus vs the C-terminus

N-terminus will represent the first translated amino acid (Met) and the C-terminus will represent the last translated amino acid

Upstream vs downstream terminology

5' -> upstream and 3' -> downstream

What direction does the ribosome move along the mRNA?

5' to 3'

What is the format for the wildtype forward primer?

it is the sequence of the top strand (the strand that is typically given to you as it is written in the 5' to 3' direction)

What is the format for the wildtype reverse primer?

it is the reverse complementary sequence of the bottom strand (the strand that you typically DO NOT see)

What does a ligase need to function?

5’ phosphate and a 3' hydroxyl

Which strand does the forward WT primer anneal to?

the bottom strand

Which strand does the reverse WT primer anneal to?

the top strand

What is true of the two mutagenic primers?

they are reverse complements of each other

What does a primer provide that allows DNA polymerase to work?

3' hydroxyl

What were the steps in purifying our plasmid which contained out gene of interest?

1. resuspension
2. lyse open the cells
3. neutralization buffer
4. purify the plasmid

What are the components of the resuspension buffer and why are they important?

Tris- pH buffer
glucose- osmotic balance
EDTA- inhibits DNAses (cut up DNA) through binding to cations which serve as cofactors for these DNAses
RNAse- degrades DNA/RNA that the bacteria makes (cleans up the gel)

What are the components of the lysis buffer?

-detergent (SDS) which breaks open the cell membrane
-base NaOH which irreversibly denatures the chromosomal DNA

What is the purpose of the neutralization buffer and what step do we use this for plasmid purification?

-we use this after lysing open the cells
-the potassium precipitates the cell debris and genomic DNA (things we don't want)
-acetate neutralizes the base NaOH that we added in the lysis buffer so that the plasmid DNA helix can renature in the supernatant

What were the two plasmids that we had to purify for our lab?

-the expression vector
-the plasmid with our gene of interest

How do we prep the expression vector plasmid after purification?

-we cut it open using restriction enzymes and then we use CIP-treatment to keep it from closing onto itself

What kind of enzyme is a restriction enzyme?

endonuclease- cuts within the DNA

What does DNA digestion refer to?

it refers to the addition of the restriction enzymes to the plasmids

What are the components of the restriction buffer used in DNA digestion?

-Mg2+ as a cofactor or the enzymes
-NaCl for ionic strength
-Tris/HCl for proper pH

Why is it important for ampicillin resistance to be included in our expression vector/plasmid?

it is advantageous for the survival of the bacteria if they take up our plasmid. Without the selectable marker, our bacteria would not take up the plasmid. Only the bacteria that have taken up our plasmid will survive once we place them on ampicillin plates

What is the purpose of CIP-treatment?

it cleaves the 5' phosphate so that ligase cannot reseal our expression vector after restriction digest

What is the purpose of the BSA in the restriction enzyme digestion reactions?

it allows for a crowding effect- some enzymes need this for optimal activity

What are the necessary components for PCR?

-template
-primers
-nucleotides
-magnesium chloride (enzyme cofactor)
-buffer with salt
-DNA polymerase

What are the main steps of PCR?

1. heat up the strands to denature them
2. annealing (temp dependent on the strand composition)
3. warm up in order to activate the polymerase which extends the primers
4. repeat

What is true of the two mutagenic primers used in site-directed mutagenesis?

they are reverse compliments

What method of transformation do we use in the lab?

calcium chloride/ heat shock (uses chemically competent cells)

What role does the calcium play in the transformation solution?

the calcium positively charged ions shield the negatively charged phosphate backbone to allow it to get across the cell membrane

What enzyme does the gene that we use for ampicillin resistance code for?

beta lactamase

What is the nutrient broth/ LB incubation phase for in terms of transformation of the plasmid?

This recovery period is to allow for the expression of beta lactamase before we put the cultures on the ampicillin plates.

How many variations/mutations of the LacI repressor did we make in the lab? What were these variants?

-there were 5 variants
-delta, WT, H17, N22, H17N22

How many variations/mutations of the LacI operator did we have in lab? What were these variants?

-there were 4 variants
-WT, T4, T6, and T4T6

How does the reporter plasmid help us determine if the LacI gene has been read?

-if the LacI gene has been read, then the lac repressor has been transcribed and translated and will bind to the operator on the reporter plasmid and prevent the beta galactosidase from being expressed so no reaction will be catalyzed (the solution will remain clear)
-if the LacI gene has NOT been read, then the repressor protein will not bind to the operator on the reporter plasmid and therefore beta galactosidase will be expressed and a reaction will occur that will convert the solution from clear to yellow (ONPG to ONP)

What gene is the LacI operator on the reporter plasmid in front of?

beta galactosidase

What is different between the two plasmids used (our plasmid with our gene of interest and our reporter plasmid) in terms of the main gene being read?

-on the LacI/AmpR plasmid, the LacI is constitutively active meaning that it has no promoter/operator and is always turned on
-on the reporter plasmid, the B galactosidase gene is controlled by an operator

Why did we use SDS solution and chloroform in the B-Galactosidase assay procedure?

we needed to pop open the cells and both these components accomplish this

What components do we add to the microfuge tubes for the B-Galactosidase assay?

-the cells
-Z-buffer
-SDS solution
-chloroform

What is a suppressor pair repression?

this is when a mutated repressor binds a mutated operator- function has been restored

What is another name for the CIP treatment enzyme?

alkaline phosphatase

What is the purpose of site directed mutations?

To observe how the functionality of a protein is affected by a nucleotide change

How do we purify the plasmid?

Affinity chromatography

How does each restriction enzyme know where to cut?

They recognize a palindromic sequence, which cuts after the 1st nucleotide

What does the restriction buffer provide?

optimal conditions

What happens if the temperature is too hot or too cold?

too hot= enzyme may be denatured

too cold= enzyme activity lowered because it limits the number of collisions, which requires a longer digestion time

Why do we use an expression vector?

We just want our gene to be amplified and we don’t want our protein altered at all so we want to preserve purity and functionality