What is transformation
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What is Transformation?. Change… In molecular biology, change in which genetic material carried by an individual cell is altered by incorporation of foreign DNA. Objective 1 - Define the term plasmid and explain its significance for bacteria and recombinant technology.

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What is Transformation?

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What is transformation

What is Transformation?

Change…

In molecular biology, change in which genetic material carried by an individual cell is altered by incorporation of foreign DNA


What is transformation

Objective 1 - Define the term plasmid and explain its significance for bacteria and recombinant technology.

A plasmid is a small piece of circular DNA in bacteria in addition to bacterial chromosome

Typically contain genes for traits beneficial for survival such as antibiotic resistance

In nature, bacteria transfer these plasmids back and forth

Acts as a vector – a gene taxi to get new DNA into bacteria


Constructing a plasmid

Constructing a Plasmid

  • Specific fragments of DNA may be isolated, cut into discrete pieces by the action of restriction enzymes, and rejoined by the action of DNA ligase

    http://www.dnai.org/b/index.html


Objective 2 describe how genomic and plasmid dna can be isolated from cells

Objective 2 - Describe how genomic and plasmid DNA can be isolated from cells.

How is DNA isolated?

http://www.iupui.edu/~wellsctr/MMIA/isolating_dna/dna_isolation_rev.swf


What is transformation

Objective 3 - Explain how the creation of sticky ends by restriction enzymes is useful in producing a recombinant DNA molecule.

Also called restriction endonucleases

1962: “molecular scissors” discovered in in bacteria

E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA

3,000 enzymes have been identified, around 200 have unique properties, many are purified and available commercially


Restriction endonucleases

“Able was I, ere, I saw Elba”

5’-GGATCC-3’

3’-CCTAGG-5’

Bam H1 site:

Restriction Endonucleases

Recognition sites have symmetry (palindromic)


Restriction enzymes

Restriction Enzymes

Enzymes recognize specific 4-8 bp sequences

Some enzymes cut in a staggered fashion - “sticky ends”

EcoRI 5’…GAATTC…3’

3’…CTTAAG…5’

Some enzymes cut in a direct fashion – “blunt ends”

PvuII 5’…CAGCTG…3’

3’…GTCGAC…5’


Why don t bacteria destroy their own dna with their restriction enzymes

Why don’t bacteria destroy their own DNA with their restriction enzymes?

Methylation!!


Why are sticky ends better

Why are sticky ends better?

  • The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other fragments.

  • Blunt ends do not have the “overlap” to bond with.


What is transformation

Complementary base pairing

5’-A-C-G-G-T-A-C-T-A-GA-A-T-T-C-A-G-C-T-A-C-G-3’

3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5’

+ DNA Ligase, + rATP

5’-C-G-G-T-A-C-T-A-G-OH

3’-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4

5’-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3’

3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5’

recombinant DNA molecule

Sticky end restriction enzymes in transformation

Human DNA cleaved with EcoRICorn DNA cleaved with EcoRI

+

PO4-A-A-T-T-C-A-G-C-T-A-C-G-3’

HO-G-T-C-G-A-T-G-C-5’


What is transformation

Restriction enzyme animation

http://www.dnai.org/b/index.html

Go to Techniques – cutting and pasting


What is transformation

Recombinant

Plasmid

E. coli host cell

Transformed Bacteria

Objective 4 - Explain each of the steps taken to isolate a gene of interest and insert it into bacterial DNA to produce the desired protein.

http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html


Making bacterial clones

Making bacterial clones

The genomic DNA and a bacterial plasmid are isolated

Both are digested with the same restriction enzyme

The fragments are mixed, and DNA ligase is added to bond the fragment sticky ends

The solution with the transformed plasmid is added to bacteria.

Bacteria take up plasmid

Bacteria are grown in culture dishes, creating “clones” that should all have the plasmid in them

But how do we check?


Objective 5 identify ways to determine if genetic transformation has been successful

Objective 5 - Identify ways to determine if genetic transformation has been successful.

A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene

The plasmid may also incorporate an ampicillin resistance gene, then the bacteria grown in the presence of ampicillin, only the bacteria with the plasmid of interest should survive.

A gene for fluorescent protein may be incorporated into the plasmid with the gene of interest, showing which organisms now carry the gene of interest


What is transformation

Ampicillin Resistance Gene

Bioluminescence Gene

pGreen Plasmid

Bioluminescent AND antibiotic resistant


Objective 6 describe how newly formed protein is isolated from transformed bacterial cells

Objective 6 - Describe how newly formed protein is isolated from transformed bacterial cells.

Wash cells from plates and centrifuge, discard liquid

Lyse bacterial cells in a non-denaturing environment (protease inhibitors and buffer solution)

Centrifuge and collect the liquid for soluble proteins and the pellet for non soluble protein

Check with gel electrophoresis


Objective 7 relate bacterial transformation to real life applications used by society

Objective 7 - Relate bacterial transformation to real life applications used by society.

Information from (among others) microvet.arizona.edu/Courses/MIC205/10GenEng/GeneticEng

Ineering and www.udel.edu/chem/bahnson/chem527

Insulin

Growth hormone

Interferon


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