CHANGING THE LIVING WORLD

How we change the living world…Selective breeding: crossing organisms with desired traits to produce the next generation.

How we change the living world…Hybridization: crossing dissimilar organisms to get the best of both.

How we change the living world…

Inbreeding: continually breeding individuals with similar characteristics.

GENETIC ENGINEERING

Genetic engineering vocab– Recombinant DNA- nucleotide sequences from two

different sources to form a single DNA molecule.

– Transgenic organism – contains a gene from another organism, typically a different species

– Genetically modified organisms (GMOs)- organisms that have acquired one or more genes by artificial means.

Figure 12.1

Genetic Engineering

Genetic engineering: The process of manipulating genes for practical purposes.

Genetic engineering may involve building recombinant DNA

DNA made from two or more different organisms.

Steps in a Genetic Engineering Experiment

Step 1 Isolate Target DNA and plasmid and cut with restriction enzymes

Step 2 Recombinant DNA is produced.

Step 3 Gene cloning: the process by which many copies of the gene of interest are made each time the host cell reproduces.

Step 4 Cells undergo selection and then are screened.

Steps in a Genetic Engineering Experiment

Step 1 The DNA from the organism containing the gene of interest and the vector are cut by restriction enzymes.

A vector is an agent that is used to carry the gene of interest into another cell

Commonly used vectors: viruses, yeast, and plasmids.

circular bacterial DNA

Plasmids

Bacterialchromosome

Remnant ofbacterium

Co

lori

zed

TE

M

Figure 12.7

Plasmid

Bacterial cell

Isolateplasmids.

Figure 12.8-1

Plasmid

Bacterial cell

Isolateplasmids.

DNA

IsolateDNA.

Cell containingthe gene of interest

Figure 12.8-2

Plasmid

Bacterial cell

Isolateplasmids.

DNA

IsolateDNA.

DNA fragmentsfrom cell

Cut both DNAswith sameenzyme.

Gene ofinterest

Othergenes

Cell containingthe gene of interest

Figure 12.8-3

Plasmid

Bacterial cell

Isolateplasmids.

Gene of interest

Recombinant DNA plasmids

DNA

IsolateDNA.

DNA fragmentsfrom cell

Cut both DNAswith sameenzyme.

Gene ofinterest

Othergenes

Mix the DNAs andjoin them together.

Cell containingthe gene of interest

Figure 12.8-4

Plasmid

Bacterial cell

Isolateplasmids.

Recombinant bacteria

Gene of interest

Recombinant DNA plasmids

Bacteria take up recombinant plasmids.

DNA

IsolateDNA.

DNA fragmentsfrom cell

Cut both DNAswith sameenzyme.

Gene ofinterest

Othergenes

Mix the DNAs andjoin them together.

Cell containingthe gene of interest

Figure 12.8-5

Plasmid

Bacterial cell

Isolateplasmids.

Clone the bacteria.

Recombinant bacteriaBacterial clone

Gene of interest

Recombinant DNA plasmids

Bacteria take up recombinant plasmids.

DNA

IsolateDNA.

DNA fragmentsfrom cell

Cut both DNAswith sameenzyme.

Gene ofinterest

Othergenes

Mix the DNAs andjoin them together.

Cell containingthe gene of interest

Figure 12.8-6

Plasmid

Bacterial cell

Isolateplasmids.

Find the clone withthe gene of interest.

Clone the bacteria.

Recombinant bacteriaBacterial clone

Gene of interest

Recombinant DNA plasmids

Bacteria take up recombinant plasmids.

DNA

IsolateDNA.

DNA fragmentsfrom cell

Cut both DNAswith sameenzyme.

Gene ofinterest

Othergenes

Mix the DNAs andjoin them together.

Cell containingthe gene of interest

Figure 12.8-7

Plasmid

Bacterial cell

Isolateplasmids.

Some usesof genes

Gene for pestresistance

Gene fortoxic-cleanupbacteria

Genes may beinserted intoother organisms.

Find the clone withthe gene of interest.

The gene and proteinof interest are isolatedfrom the bacteria.

Clone the bacteria.

Recombinant bacteriaBacterial clone

Gene of interest

Recombinant DNA plasmids

Bacteria take up recombinant plasmids.

Harvestedproteins may beused directly.

Some usesof proteins

Protein for“stone-washing”jeans

DNA

Cell containingthe gene of interest

Protein fordissolvingclots

IsolateDNA.

DNA fragmentsfrom cell

Cut both DNAswith sameenzyme.

Gene ofinterest

Othergenes

Mix the DNAs andjoin them together.

Figure 12.8-8

RESTRICTION ENZYMESmolecular scissors

A Closer Look: Cutting and Pasting DNA with Restriction Enzymes

– Recombinant DNA is produced by combining two ingredients:

A bacterial plasmid The gene of interest

How do we cut them?

• Using restriction enzymes: bacterial enzymes which cut DNA at specific nucleotide sequences

• produce pieces of DNA called restriction fragments.

• Why do you think bacteria contain restriction enzymes?

Restriction Enzymes are palindromes: the same forward as backwards, like RACECAR.

Examples:

GAATTC CCCGGG AAGCTTCTTAAG GGGCCC TTCGAA

G AATTC CCC GGG A AGCTTCTTAA G GGG CCC TTCGA A

Sticky EndsBlunt End

Recognition sequencefor a restriction enzyme

Restrictionenzyme

Sticky

end

Stickyend

DNA

A restriction enzyme cutsthe DNA into fragments.

Figure 12.9-1

Recognition sequencefor a restriction enzyme

Restrictionenzyme

Sticky

end

Stickyend

DNA

A DNA fragment is addedfrom another source.

A restriction enzyme cutsthe DNA into fragments.

Figure 12.9-2

Recognition sequencefor a restriction enzyme

Restrictionenzyme

Sticky

end

Stickyend

DNA

A DNA fragment is addedfrom another source.

A restriction enzyme cutsthe DNA into fragments.

Fragments stick together bybase pairing.

Figure 12.9-3

DNA LIGASE –DNA ligase connects the DNA fragments into one continuous strand (DNA Glue or tape)

Recognition sequencefor a restriction enzyme

Restrictionenzyme

Sticky

end

Stickyend

DNA

DNAligase

Recombinant DNA molecule

A DNA fragment is addedfrom another source.

A restriction enzyme cutsthe DNA into fragments.

Fragments stick together bybase pairing.

DNA ligase joins thefragments into strands.

Figure 12.9-4

Recognition sequences

DNA sequence

Restriction enzyme EcoRI cuts the DNA into fragments.

Sticky end

Your turn to try!!

– Plasmids:• Can easily incorporate foreign DNA

• Are readily taken up by bacterial cells

• Can act as vectors, DNA carriers that move genes from one cell to another

• Are ideal for gene cloning, the production of multiple identical copies of a gene-carrying piece of DNA

Bacterial cells don’t edit the RNA, so how can they make the correct protein?

Genetic Engineers can eliminate the introns from mRNA and reverse the process, producing a DNA strand that is only the instructions for the protein.

Use Reverse Transcriptase

Cell nucleus

DNA ofeukaryoticgene

Test tube

Transcription

Exon Intron Exon ExonIntron

Figure 12.11-1

Cell nucleus

DNA ofeukaryoticgene

RNAtranscript

mRNA

Test tube

Transcription

Introns removed andexons spliced together

Exon Intron Exon ExonIntron

Figure 12.11-2

Cell nucleus

DNA ofeukaryoticgene

RNAtranscript

mRNA

Test tube

Reversetranscriptase

Transcription

Introns removed andexons spliced together

Isolation of mRNA fromcell and addition ofreverse transcriptase

Exon Intron Exon ExonIntron

Figure 12.11-3

Cell nucleus

DNA ofeukaryoticgene

RNAtranscript

mRNA

Test tube

Reversetranscriptase

cDNA strandbeing synthesized

Transcription

Introns removed andexons spliced together

Isolation of mRNA fromcell and addition ofreverse transcriptase

Synthesis of cDNAstrand

Exon Intron Exon ExonIntron

Figure 12.11-4

Cell nucleus

DNA ofeukaryoticgene

RNAtranscript

mRNA

Test tube

cDNA of genewithout introns

Reversetranscriptase

cDNA strandbeing synthesized

Transcription

Introns removed andexons spliced together

Isolation of mRNA fromcell and addition ofreverse transcriptase

Synthesis of cDNAstrand

Synthesis of second DNAstrand by DNA polymerase

Exon Intron Exon ExonIntron

Figure 12.11-5