R.O Lecture 5

Question 1

What validation methods can be used to determine if DNA cloning was successful?

Answer 1

1.Selectable markers
2.Blue/white colony screening
3.Restriction enzyme digest
4.Colony PCR
5.Sequencing
6.Recombinant protein detection and analysis

Question 2

1. Selectable Markers

Answer 2

Selectable markers are often antibiotic resistance genes. They help in artificial selection. They help in identifying transformed cells and selectively allowing their growth whereas eliminating non-transformants in cloning vector.

Question 3

1. What type of genes are used as selectable markers?

Answer 3

Can be antibiotic resistance genes e.g. ampr , tetr
Encode proteins that make the cell resistant to relevant antibiotics

Question 4

ampr

Answer 4

ampr : encodes enzyme that hydrolyses active portion of ampicillin

Question 5

tetr

Answer 5

tetr : encodes efflux pump that removes tetracycline from the cell

Question 6

How do we know that the plasmid has been taken up by the cells? (selectable marker)

Answer 6

Select for transformed cells: Only bacteria that take up the plasmid with the selectable marker will be able to grow in the presence of the antibiotic

Question 7

What method is used to insure that the “DNA of interest” has been succesfully inserted into the plasmid at the MCS?

Answer 7

Blue/white colony screening

Question 8

What vector is blue/white colony screening used for?

Answer 8

Used in DNA cloning not expression plasmids.

Question 9

What gene is used in Blue/White colony screening?

Answer 9

lacz gene

Question 10

What enzyme does the lacZ gene encode for?

Answer 10

*MCS is located within the
lacZ gene on the plasmid

*lacZ gene encodes the β galactosidase enzyme;
under the control of the lac promoter

Question 11

What does the βgalactosidase enzyme do?

Answer 11

*β galactosidase cleaves lactose into glucose and
galactose

*β-galactosidase is made up of two fragments, alpha and omega
When the two fragments are associated they form functional enzyme

*It Converts substrates such as X-gal (5-bromo-4-chloro-3-indolyl-[beta]-Dgalactopyranoside) a colourless modified galactose sugar into a blue coloured product

Question 12

How are white colonies formed?

Answer 12

If a DNA insert is cloned into the MCS within the lacZ gene, the gene will be
disrupted, β-galactosidase will not be produced, and X-gal will not be
metabolised (white colonies).

Question 13

IPTG

Answer 13

IPTG (Isopropyl β-D-1-thiogalactopyranoside) is a molecular mimic of allolactose, a lactose
metabolite that triggers transcription of the lac operon: used to induce expression of inserted gene

Question 14

What do blue and white colonies represent?

Answer 14

Blue colonies therefore show that they may contain a vector with an uninterrupted lacZα (therefore no insert), while white colonies, where X-gal is not hydrolyzed, indicate the presence of an insert in lacZα which disrupts the formation of an active β-galactosidase.

Question 15

Diagnostic Restriction Enzyme Digest

Answer 15

Using restriction enzymes, can perform a diagnostic digest of plasmid to determine if DNA insert has been incorporated

Fragments generated will be different sizes depending on
whether the DNA insert is present or not

Question 16

What does Diagnostic Restriction Enzyme Digest detect?

Answer 16

Analyse the digested plasmid on an agarose gel to determine if generated fragments are the correct size.

Can also determine if the DNA insert is in the correct
orientation in the MCS

Question 17

What does Colony PCR Detect?

Answer 17

Detects presence/orientation of DNA insert using
primers designed to amplify a region of the
insert/plasmid

Question 18

How is Colony PCR carried out?

Answer 18

• Transformed bacteria are spread on agar
  plates to isolate individual colonies.
• Primers are designed to anneal to the
  insert/plasmid (green, blue, red arrows).
• PCR is carried out using DNA from each colony
• PCR product is analysed by gel electrophoresis
  to determine if it matches predicted size.

(look at slide 7 for diagram)

Question 19

Sequencing

Answer 19

Sequencing the recombinant plasmid is the most accurate way to determine if insert has been incorporated correctly

Can determine if:
- the insert is in the correct orientation within the plasmid
- the inserted gene is in frame for correct translation
- the gene sequence is the same as reported in the genomic database

Question 20

What are the methods for sequencing?

Answer 20

• Sanger sequencing (uses fluorescent dideoxynucleotides)
• Next-generation sequencing (for larger sequences)

Question 21

What method is used to verify if the inserted “gene of interest” produce the expected protein product?

Answer 21

Recombinant protein production and analysis

Question 22

Describe Recombinant protein production and analysis.

Answer 22

Following cloning of a gene into an expression plasmid and transformation
into a host system, protein expression can be induced and the protein of
interest can be purified and characterised.

Peptide tags (e.g. 6xHis Tag) or fusion
proteins (GST) can enable detection
and purification recombinant protein.

Question 23

What are the steps in recombinant protein purification?

Answer 23

1. Design expression plasmid, transform, select
2. Grow culture of positive clone*, induce expression
3. Lyse cells
4. Isolate protein-containing fraction (centrifugation, protein solubilisation)
5. Purify from contaminating proteins (Column Chromatography—collect fractions)
6. Assess purity on SDS-PAGE (Detect with antibodies etc.)

Question 24

What can be used to identify positve clones?

Answer 24

Verification methods discussed previously can be used to identify
positive clones containing the ‘gene of interest’

Question 25

Why is Cell Lysis required? Name the methods of Cell Lysis

Answer 25

Cell Lysis is required to extract the cell.

Lysozyme, Sonication, High-pressure homogeniser and Bead-miling

Question 26

Lysozyme

Answer 26

• enzymatic breakdown of E. coli cell wall
• 1 hr at 4°C

Question 27

Sonication

Answer 27

Uses sound waves to aid cell lysis

Question 28

High-pressure homogeniser

Answer 28

Physical method of cell lysis

Question 29

Bead-miling

Answer 29

Cells disrupted by coliding with beads.

Question 30

What is used to inhiibt proteolysis and denaturation? (Cell disruption)

Answer 30

In order to inhibit proteolysis and denaturation, protein purification is usually
carried out
- In the cold (on ice or in a cold room)
- In the presence of protease inhibitors (small molecules that inhibit
specific proteases)
- Metal chelators (EDTA, EGTA) inhibit metalloproteases

Question 31

Inclusion Bodies

Answer 31

Bacteria overproducing recombinant proteins are subjected to different stresses - metabolic and confrontational.

protein processing associated with cell stress responses might
render useless products
- mainly because of lack of solubility, and many protein species deposit in high amounts as protein aggregates known as inclusion bodies (IBs)

Question 32

What do inclusion bodies contain?

Answer 32

• recombinant protein in variable proportions (from 60 to more than 90%)
• some contaminants as chaperones, DNA, RNA and lipids

Question 33

Eukaryotic proteins in IBS

Answer 33

Many eukaryotic proteins are exclusively found trapped in IBs and seem to be
resistant to process-based solubility enhancement

Question 34

Recombinant proteins that have been succesfully purified from IBs

Answer 34

Betaferon and insulin

Question 35

Non Intergral membrane proteins from IBs

Answer 35

For non integral membrane proteins, cytosolic and/or soluble protein domains, the
probability of success is reasonably high

Question 36

How to Purify proteins

Answer 36

*Chromatography
It can separate proteins based on
differences in charge, size, or affinity for
other molecules/ligands.

(slide 11)

Question 37

Affinity Chromatography

Answer 37

Makes use of specific binding interactions between molecules

Question 38

Affinity Chromatography Process

Answer 38

1. Incubate crude sample with the immobilized ligand.
2. Wash away non bound sample components from solid suuport
3. Elute

Question 39

What the are the commonly used affinity columns?

Answer 39

• Ni2+ → binds to poly Histidines (example 6xHis)
• Specific antibodies (anti-FLAG tag)
• glutathione → binds to GST (Glutathione S-transferase)
• Calmodulin → binds to CBP (Calmodulin binding protein)
• Protein A or G → binds antibodies

Question 40

What is the high affinity of the Ni-NTa resins for GxHis-tagged proteins or peptides due to?

Answer 40

1. the strength with which these ions are held to the NTA resin
2. the specificity of the interaction between histidine residues and
   immobilized nickel ions