Labs Flashcards
(47 cards)
Define pUC derived
plasmid present in e.coli
Define T7promoter
Located downstream from FP promoter, allows T7 RNA polymerase in cell to maintain continous expression
Define 6xHis tag
A poly-histidine, tag provides metal binding site for purification through NTA-chelate affinity chromatography
Where is the 6xHis tag found
In frame with a sequence that includes; N terminal fusion peptide, initiation codon. All located upstream from the fluorescent protein
What is the ori-locus
Origin of replication
What does the ori-locus do
Allows plasmid to be independently replicated in host cell.
What is the importance of ampicillin resistance in the experiment
Allows transformed cells to reproduce in presence of ampicillin which destroys e.coli cells ability to create cell walls. Meaning only successful cells would grow.
Define transformation
Inserting foreign plasmids into a cell to alter the genetic makeup
Define plasmid
Circular strands of DNA from cytoplasm of bacteria and yeast cells usually
Define cloning vector
Plasmids modified to include foreign DNA that can be expressed in bacteria to produce proteins within the vector
Where does the DNA for plasmids come from
Fungi, Bacteria, Plants, animals
Why is transformation important
Useful for genetic engineering because it acts like a virus and can create specialized proteins
How is insulin made
Transformation
Define competence, and how it is achieved in this lab
Some cells that cannot pick up plasmids naturally must be made competent to absorb it, this lab uses chemical transformation.
Define neutralization of charges and explain how it was achieved
Calcium cations in a solution can neutralize the negative charges in the cell membrane and DNA. Calcium chloride and heat shock were used to make the cells competent, then they were placed on ice in a calcium solution to be neutralized.
Why was neutralization of charges done on ice
The cold slowed down membrane fluidity to make a more efficient neutralization
Define heat shock
A sudden increase and subsequent decrease in temperature, causing a change in pressure between the insideq and outside of the cell, forcing the plasmid in through the pores
How is it ensured that the colony is identical
One bacterial cell performer binary fission to produce the entire colony
Briefly explain how cell extract was prepared
1 isolated colony was inoculated in LB and ampicillin solution
The cells were allowed to grow as they are incubated under agitation
After cell growth cell membranes are broken with a lysis treatment and a detergent which release cytoplasm
what detergent is used in this lab
SDS
What is LB
A nutritious broth to aid growth
What does agitation do
Provides cells with oxygen, causing faster growth and division
Briefly explain NTA-Chelate affinity chromatography
The 6x-His tag in fluorescent proteins binds to nickel in the nickel charged affinity resin, which prevents them from washing away like non fluorescent proteins
4 steps for NTA-Chelate affinity chromatography
- Lysis of membrane
- Addition of nickel charged resin
- Washing resin for non bound components
- Elution of protein using elution buffer
