Biotechnology lab 2

Question 1

What is the objective of this lab?

Answer 1

Induce the expression of a recombinant protein in E. coli.

Question 2

What is the first step to induce the expression of a recombinant protein?

Answer 2

The construction of a functional gene expression vector.

Question 3

What is the most popular vector?

Answer 3

A plasmid.

Question 4

What is a plasmid?

Answer 4

A circular DNA molecule that can replicated independently from the host chromosome.

Question 5

How is a plasmid generally introduced?

Answer 5

Through transformation.

Question 6

In this lab, how was a recombinant expression vector constructed?

Answer 6

By inserting the DNA sequence of the human PTEN into a pET vector.

Question 7

What does the cloning vector pET contain?

Answer 7

Both the transcriptional and translational elements necessary to regulate the gene expression of PTEN.

Question 8

What does the pET-PTEN expression vector contain?

Answer 8

A well characterized promoter located upstream of the multiple cloning site. It specifically contains: an ampicillin resistance marker, ColE1 origin of replication, F1 origin of replication, lacI gene, T7 transcription promoter, Lac operator region upstream of the promoter, Multiple cloning site where PTEN was inserted, 6x His tag.

Question 9

Where was PTEN inserted in the expression vector?

Answer 9

In the multiple cloning site.

Question 10

What is the purpose of the 6xHis tag?

Answer 10

To allow purification of the target protein utilizing affinity chromatography.

Question 11

Where is the 6x His tag located?

Answer 11

At the N-terminus of the PTEN sequence.

Question 12

Where are the transformed bacteria grown?

Answer 12

In liquid media containing ampicillin.

Question 13

Which cells will be able to survive the ampicillin media?

Answer 13

Only cells containing the PTEN plasmid which expresses B-lactamase, allowing for ampicillin resistance will be able to propagate.

Question 14

What do the two origins of replication allow?

Answer 14

For multiple copies of the vector to be produced.

Question 15

What does the genetic engineering of the vector so it contains both the lac promoter and operator allow?

Answer 15

Allow the artificial induction of gene expression by the addition of a lactose analogue, IPTG, which binds to lacI (the lac repressor (transcriptional)) to allow RNA polymerase access to the promoter.

Question 16

What is the T7 transcriptional promoter designed to be specific to?

Answer 16

The T7 RNA polymerase.

Question 17

What is the disadvantage of using a bacterial host system?

Answer 17

The lack of a mammalian post-translational modification system that may be imperative to protein folding and activity.

Question 18

In this lab, we will be exploiting the bacterial expression system in E. coli to produce what?

Answer 18

Large quantities of PTEN.

Question 19

What is the bacterial cell line that will be used?

Answer 19

BL21 (DE3)-Codon plus-RIL competent cell line.

Question 20

What is special about this cell line?

Answer 20

They contain additional copies of rare tRNAs that when lacking, can normally limit the translation of proteins in conventional E.coli strains.

Question 21

What do the argU, ileY annd leuW tRNA genes encode?

Answer 21

tRNAs that recongize arginine, isoleucine and leucine codons enabling for improved heterologous protein production that may be otherwise restricted in A-T rich genomes.

Question 22

What do the Ion and ompT proteases do?

Answer 22

Degrade proteins during recombinant purification.

Question 23

Why do we need to use the Victor plate reader?

Answer 23

Use to measure the absorbance which is used to determine if the bacteria are in the log state of growth.

Question 24

What is the point of the ColE1-compatible pACYC?

Answer 24

confers resistance to chloramphenicol which is required in the medium to maintain the pACYC plasmid

Question 25

What does the endonuclease 1 (endA) do?

Answer 25

degrades DNA in miniprep procedures.

Question 26

What reagent is used to clean the sonic micro tip?

Answer 26

Ethanol

Question 27

What is done to separate the soluble proteins from the non-soluble ones?

Answer 27

centrifugation at 20000g in a pre chilled (4 degrees C) floor mounted centrifuge for 15 minutes.