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Flashcards in Biotech Final - Lab 2 Deck (18)
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What was the objective of lab 2?

To artificially induce the expression of a recombinant protein in E. coli.


What is a plasmid?

Circular ds DNA molecule that has the ability to replicate independently of the host chromosomes.


How is a plasmid introduced?

Generally by transformation (although transduction and other methods are available).


What vector was PTEN inserted into?

pET vector.


What does the pET plasmid contain?

1) MCS
2) Lac promoter and upstream lac operator region
3) Ampicillin and chloremphenicol resistance markers
4) ColE1 and F1 origins of replication
5) LacI gene
6) T7 phage promoter
7) PTEN fusion protein sequence containing a His6 tag fused to the N-terminus


Why are transformed bacterial cells grown in media containing antibiotics?

Only cell containing the PTEN plasmid (that expresses B-lactamase) will be able to propagate. (Ampicillin is part of the B-lactam family of antibiotics)


What was done to determine what phase of bacterial growth the cells were in?

Take readings of blank media and that containing the transformed cells. A victor plate reading of 0.6 indicates a density in the log phase.


What is added to transformed E. coli cells?

IPTG, ampicillin and chloramphenicol


Protein isolation requires what?

Lysis buffer and sonication.


What does the lysis buffer contain?

Protease inhibitors.


Why are protease inhibitors important?

When the membrane of a cell is lysed, endogenous protease are released and will degrade proteins of interest unless inhibited.


What is sonication?

Physical disruption used to break open cells. The method uses pulses of high frequency sounds waves to agitate and lyse cells. A probe delivers these soundwaves when immersed in solution. This is often coupled with a lysis solution containing protease inhibitors.


Why are samples spun in a pre-chilled floor-mounted centrifuge? (Prior to lysis)

To pellet cells and allow for removal of media.


How is the pellet resuspended?

Using lysis buffer.


What is the Bradford Assay?

A spectroscopic analytical procedure used to measure the concentration of protein in a solution.


What is the Bradford assay based on? (Hint: colometric protein assay)

The absorbance shift of the Coomassie Brilliant Blue dye. In acidic conditions, the red anionic form of the dye is converted to its bluer form to bind to the protein being assayed.


This assay is susceptible to interference by what?

SDS and similar detergents


Why is the sonicated sample centrifuged?

Separate soluble proteins from non-soluble cellular debris.

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