Laboratory: General Information Flashcards
What are plasmid vectors?
(7)
Tiny extra-chromosomal pieces of double stranded circular DNA
Found in bacterial cells
Couple thousand base-pairs long
Many copies per cell
Autonomously self replicating (have own origin or replication)
Carry genes that convey antibiotic resistance
Have restriction enzyme cut sites
What type of genes do plasmids usually have?
Antibiotic resistance genes
Plasmids have restriction enzyme cut sites what is this useful for?
The uptake of foreign DNA
What is an operon system?
Genes that work together and are regulated together
Give three examples of operons
Lac operon
Trp operon
Arabinose operon
List the different parts
(6)
Bacterial origin of replication
Selectable marker e.g. Ampicillin resistance
Multiple cloning site
Strong inducible, promoter with control e.g. Lac
Methods of inhibiting protein reduction e.g. LacI
Method of purifying protein (GST, His fusions)
TE buffer-Tris EDTA buffer
(4)
It is a buffer used to protect DNA from degradation
It is used to store DNA
The Tris, CI pH 8 in buffer is to maintain the PH of solution in alkaline
The EDTA in TE buffer is a chelating agent. It chelates Ca and Mg ions which are cofactors required by DNAses
Write a note on the arabinose operon
(4)
The arabinose operon uses both positive and negative control
Contains three different genes (ara A, ara B and ara D) which code for three enzymes needed to convert arabinose to a usable form
A fourth gene codes for a protein (ara C) that acts to regulate the structural genes
They encode metabolic enzymes for breaking down arabinose
What is the ampicillin resistance gene called?
bla
What is a pGLO plasmid made of?
(4)
Green fluorescent protein gene
araC gene (regulates GFP transcription)
bla gene (ampicillin resistance gene) ( produces beta-lactamase enzyme)
ori (origin of replication - allows plasmid replication)
What does the bla gene produce?
Beta lactamase
Which induces ampicillin resistance
What does the bla gene produce?
Beta lactamase
Which induces ampicillin resistance
How does transformation take place?
Treat bacteria with calcium chloride and then undergo heat-shock @42 degrees celsius
How does transformation via Calcium chloride work?
The positive charge of calcium chloride ions neutralises:
- the negative charge of DNA phosphates
- the negative charge of membrane phospholipids
- slows down the fluidity of the membrane and cell wall
How does heat shock work?
Activates the heat shock proteins and seals the cell membrane and traps plasmid inside
What do you do after plasmid transformation?
Recovery in highly rich nutrient broth (SOC medium)
This allows the bacteria to recover from the transformation procedure and allows the b-lactamase gene to produce ampicillin to facilitate the selection process
How have we modified the araC gene?
(3)
AraC is now the regulator for GFP
The genes of the ara operator (B, A and D) have been replaced with the gene for GFP
Now the addition of arabinose turns the araC operon on which thusly expresses GFP
What does the araC gene usually do?
The araC gene or arabinose operator is responsible for breaking down arabinose into usable parts
The B, A and D genes of araC are responsible for making enzymes
What happens before and after you introduce arabinose to the modified araC gene?
(3)
Without arabinose the araC repressor blocks transcription of the gene and therefor GFP
In the presence of arabinose, arabinose binds repressor and changes its conformation
The altered repressor leaves DNA and now RNA polymerase can perform transcription and express the GFP
What is electroporation?
Exposing cells to high voltage in the presence of plasmid DNA and low salt conditions
Allows for uptake of plasmid
