chapter 20 - dna technology and genomics genetically modify organisms and transgenic organisms...

Chapter 20 - DNA Technology and Genomics

Genetically modify organisms and transgenic organisms

Genetically modified organisms (GMO’s):

-Organisms whose genes have been altered using genetic engineering techniques.

- Most GMO’s are transgenic organisms… they have received genes from a different organism.

Transgenic organisms

Ex. A mouse is given a gene from a human. The mouse is a transgenic GMO.

Trans- ; across (across species in this case)


Genetically modify organisms (GMO’s) and transgenic organisms

GloFish1. Zebra danio was genetically engineered with a gene from sea coral that causes the fish to glow in the presence of environmental toxins.2. Gene was inserted into the embryo of the fish.

Zebra danio

3. First GMO available as a pet.

GMO’s at home:



GFP Mice

GFP (green fluorescent protein)

Aequorea victoria (jellyfish, phylum cnidaria)

1. Gene from a jellyfish (Aequorea victoria) that codes for GFP was inserted into the embryos of mice.

GMO’s in research:



GFP (green fluorescent protein) – a reporter protein

1. GFP is used in cellular and molecular biology.2. You can attach this protein to any other protein you want making it a reporter protein.

- It “reports” to you where the protein is going since it emits green light (similar to radioactivity in that sense)




Ex. - GFP has been attached to a protein called MFD, which is found in peroxisomes.

- Those little green dots are peroxisomes…- You can track any protein you want…in a single cell or an entire organism




1. Corn plants containing Cry genes from a bacterium – Bacillus thurengensis.2. The genes code for enzymes that produce a toxin (insecticide), Bt toxin, which will kill European corn borer larvae, the most damaging insect to corn in US and canada.

Bt Corn European Corn Borer Larva

GMO food:



1. Corn plants containing Cry genes from a bacterium – Bacillus thurengensis.

Are these toxins safe for you to eat???

Bt Corn European Corn Borer Larva

GMO food:



Ordinary rice“Golden” rice

- “Golden” rice is genetically engineered with genes that code for enzymes that make beta-carotene, a precursor to Vitamin A for countries deficient in foods with Vit. A…- This rice has never been used because of environmental concerns.

GMO food:



Genetically engineered sheep with the human gene for alpha-1-antitrypsin (AAT). AAT is extracted from their milk and used to treat humans deficient in AAT, which is one cause of emphysema (a breathing disorder) in approximately 100,000 people in the western world.

AAT Sheep

GMO medicine:



- Insulin is made using the bacterium E. coli.

- The human gene coding for insulin is inserted into E. coli, which will then make insulin for us (we will see how this is done shortly)…

E. Coli with the human insulin gene

GMO medicine:



Conclusion- We can basically move any gene(s) between members of a species or between any species. - We can also alter the genes to our liking (GFP tagged proteins) before inserting them into embryos.

Is all of this genetic engineering positive, negative?



Let’s look at some of the ways we genetically engineer organisms starting with how we can take a human insulin gene and put it into E. coli…

Chapter 20 - DNA Technology and GenomicsHow can we use bacteria to manipulate DNA and protein?

First we must understand bacteria and how they take up DNA…

(it is more than just mutation that gives certain species of bacteria their genetic diversity)

Fig. 12.1A-C


1. Transformation

Bacteria can take up a free piece of bacterial DNA

There are three methods by which bacteria take up DNA in nature.

Griffith

Fig. 12.1A-C


2. Transduction

Bacteriophage is mistakenly packaged with bacterial DNA. Injects this DNA into another bacteria.


Hershey and Chase

Fig. 12.1A-C


3. Conjugation

“Male” (F+) bacteria extend sex pili (long tube) to “female” (F-) bacteria. Part of chromosome is replicated and transferred.


Fig. 12.1D


Once the DNA is transferred, integration must occur:

Crossing over occurs (where do you think we got it from?) and the new DNA is integrated in place of the original DNA, which is degraded.


We will focus mostly on transformation when we look at genetic engineering…

1. Transformation2. Transduction3. Conjugation


Transformation in the lab:

Heat Shock Method in bacteria

1. Transformation

1. Take bacteria in a tube (in solution)

2. Add the DNA you want it to take up into the tube.

3. Let the tube chill on ice for a few minutes4. Then quickly heat the tube to 42°C (107°F) for 90 seconds.- This will open up “holes” in the bacterial

membrane for the DNA to slip in.

5. Cool on ice for 10 minutes…done

The bacterium now has the DNA…simple.


Bacteria can have more than just a single circular chromosome…(They may have little circular extra-chromosomal

DNA called Plasmids)

extra-chromosomal = outside of the chromosome like extra-terrestrial means coming from outside Earth (E. T.)

Fig. 12.2C


The majority of the DNA above is chromosomal, but you can see the small circular pieces not part of the chromosome…plasmids.


Plasmid - Small, circular piece of DNA distinct from bacterial chromosome - has own origin of replication (ori)

- Carries assorted genes

- called vectors when used in genetic engineering…

mcs


Vectors- We have engineered plasmids to contain clusters of restriction sites called polylinker regions or multiple cloning site (mcs) where we can easily insert the gene of our choice. - We have also engineered these plasmids to contain an antibiotic resistance.

mcs


Vector Summary1. Ori (origin of replication)

3. Antibiotic resistance gene like ampr (ampicillin resistance) for selecting positive transformants.

mcs

2. MCS for inserting gene of choice

Chapter 20 - DNA Technology and GenomicsHow can we use bacteria to manipulate DNA and protein?An actual vector: pET16b

mcs

ori

Amp resistance

The bacterium has enzymes called restriction enzymes that attempt to cut up the bacteriophage DNA before it can take over the cell. Different species have evolved different restriction enzymes…

Chapter 20 - DNA Technology and GenomicsHow can we use bacteria to manipulate DNA and protein?Recall how a bacterium defends itself when a bacteriophage injects its DNA into a bacterium…

Aside: Why do these enzymes not cut the bacterial chromosome?The bacterial chromosome is methylated (modified by adding –CH3 groups so the enzymes can’t bind to it)

Restriction enzymes

1. molecular DNA scissors (enzymes that cut DNA)


2. Different restriction enzymes cut different sequences.3. Scientists have isolated hundreds of different restriction enzymes from many different bacteria – EcoRI, BamHI, NcoI, etc…

Fig. 12.4

Restriction enzymes


Ex. EcoRI

Notice anything interesting about this sequence?

- It is palindromic, read the same way forward and backward on each strand.

- Majority of restriction sites are palindromic…

Notice that is doesn’t cut straight through like paper scissors. The enzyme cuts each strand on the 3’ side of G generating single-strand regions called sticky ends.

Restriction enzymes


Ex. EcoRI

EcoRI

Why do you think we call them sticky ends?

Restriction enzymes


Ex. EcoRI

EcoRI

Because they can base pair to a complementary sticky end…they are “sticky”. If it cut straight through then it could not base pair.

More examples of restriction enzymes


Restriction enzymes


Now that we understand transformation, plasmids and restriction enzymes, we are ready to take the next step and learn how to take a gene from an organism of choice (ex. Human insulin) and put it into a bacterium so that the bacterium can make the polypeptide (ex. insulin) for us. This process is called subcloning.

Subcloning

Fig. 12.3

Chapter 20 - DNA Technology and GenomicsHow can we use bacteria to manipulate DNA and protein?General overview of gene cloning(aka subcloning)

Let’s look at how we do this…

Now imagine this restriction site was engineered into a plasmid (now called a vector) as shown above.


Ex. EcoRI

plasmid (vector)

SubcloningRestriction site engineered into the polylinker (mcs) region

What happens if you treat it with the restriction enzyme BamHI?


Ex. EcoRI

BamHI

Nothing, BamHI does not cut that sequence.

Subcloning


Ex. EcoRI

EcoRI

What happens if you treat it with the restriction enzyme EcoRI?

Subcloning

EcoRI cuts the vector leaving two sticky ends…


Ex. EcoRI

EcoRI

Now what?

Subcloning

We need to insert our gene of choice into the plasmid.

Fig. 12.3


1. You can isolate the DNA from the organism of interest, which has the gene you want to put into the vector. You will likely do this using PCR (polymerase chain reaction), a technique we will discuss later on.

Subcloning


…CGATTAGAATCCCGCC CGGATTGAATCCCGAA… …GCTAATCTTAGGGCGG GCCTAACTTAGGGCTT…

Insulin gene

Zoom in

Subcloning

What do we need to do?

Fig. 12.3


2. Cut the gene with the same restriction enzyme that you cut the plasmid/vector with to get complementary sticky ends.

…CGATTAGAATTCCGCC CGGATTGAATTCCGAA… …GCTAATCTTAAGGCGG GCCTAACTTAAGGCTT…

Insulin gene

Zoom in

Subcloning


…CGATTAG AATTCCGCC CGGATTG AATTCCGAA… …GCTAATCTTAA GGCGG GCCTAACTTAA GGCTT…

Insulin gene

Zoom in

What now?

Subcloning

3. Mix countless numbers of cut vector with countless numbers of cut gene…what should happen?


+ AATTCCGCC CGGATTG GGCGG GCCTAACTTAA

Insulin gene

Subcloning

The sticky ends should base pair (the two pieces anneal = base pair to each other).


However, you still have gaps between the nucleotides in each strand…what should we do?

Subcloning

Use DNA ligase + ATP to ligate the strands together


Every enzyme/protein we discover is a new tool for scientists to use in the lab to manipulate DNA. DNA ligase was discovered when investigating DNA replication, but now we use it as “glue” when subcloning genes into vectors.

DNA ligase

Subcloning

Now what should we do with this vector containing our gene or interest?

Put it into bacteria like E. coli by transformation using the heat-shock method.


Subcloning X 1,000,000’s

Combine millions of vector with millions of E.coli and heat shock…

One can also use electroporation (forming pores using electricity) to transform as opposed to heat shocking. It involves sending electricity through the vector/bacterial solution, which induces temporary holes in the bacterial membrane for vector to enter. This is also routinely used to “transform” eukaryotic cells. I will put a slide of this later on.



Transformation efficiency if LOW: (very few of the bacteria will receive a vector…)

How can we identify the ones that do get a vector? Remember that antibiotic resistance

gene in the plasmid?If you take these billions of bacteria and spread them on an agar plate (a sort of nutrient rich jello) containing antibiotic, only those that have the vecor (have the resistance gene) will grow...see next slide for agar plate


Subcloning

Transformation efficiency if LOW: (very few of the bacteria will receive a vector…)• This agar plate (right)

contains the antibiotic ampicillin. • The yellow dots you see are called colonies.

•Each colony arose from a single ampicillin resistant bacterium spread onto the plate the night before

•Each colony is a group of millions of bacteria that are essentially genetically identical (clones) because…

Agar Plates:

Millions of bacteria were put on this plate…how many were transformed with a vector?Count the colonies, only about 100 or so.

Bacterial colonies (yellow dots)


Subcloning-DNA means no vector transformed+DNA means vector was transformedLB = luria broth = bacterial food in agarAMP = ampicillin

Don’t worry about ARA

Here you can see that when no vector (-DNA) was used as a control in the transformation, the bacteria grew only on the LB plate, not the plate with ampicillin. What you see on the LB plate is known as a “lawn” of bacteria , which means that so many grew that all the colonies mix together and just cover the plate.

When vector was used (+DNA) bacteria that got the vector could grow on the AMP plates and colonies are observed.


Reminder: The vector has an origin of replication, and will be replicated by DNA polymerase during binary fission.

Now our gene is inside the bacteria. How does this help us?


Subcloning


1. We can take the bacteria after many round of binary fission and isolate the plasmid/vector, and take back the gene. In essence, the bacteria replicated it for us…2. Or we can have the bacterium make the protein for us and then we can take the protein and use it.

Fig. 12.3


Review Slide

We have a few more problems 1. Restriction digests are not 100% 2. Ligations have very low efficienty

Plasmid(1,000,000’s in a small volume of aqueous solution in tiny Eppendorf tube)


Restriction digest

Restriction digest

Cut plasmid

Only a fraction will get cut

Uncut plasmid

+ Insert ligatesGene insert

ligation

ligation

Plasmid self-ligates

After ligation, you have a mix of plasmid where most of it does not contain your insert. How can we identify the ones that do?

Restriction digest not 100% and Ligation efficiency is LOW:

Chapter 20 - DNA Technology and GenomicsHow can we use bacteria to manipulate DNA and protein?Ligation efficiency is LOW

The plasmid can be engineered to have a lacZ gene within the polylinker (mcs) as shown above.

How does this help?If the gene gets inserted, the lacZ gene will be non-functional. If the gene is not inserted then lacZ will be fine.

First, remember what lacZ does…It codes for the enzyme beta-galactosidase, which hydrolyzes lactose to glucose and galactose.

I still don’t see how this helps…


We have designed a small molecule called X-gal, which resembles lactose and is hydrolyzed by beta-galactosidase as shown above.So What?

OK, but how does this help you determine which bacteria contain plasmid with insert?

X-gal is clear (no color, absorbs no light), while the molecule that forms after hydrolysis is blue…


After transformation and overnight growth on agar plates containing X-gal, some colonies will turn blue meaning they have a working beta-galactosidase enzyme and therefore do not contain your gene. Those that remain white do not have the functional enzyme and therefore must have your gene.

First, the bacteria we use have a natural mutation in their genomic lacZ gene. Basically, they do not have lac Z.

BLUE-WHITE SCREENING:


Why do the agar plates contain the antibiotic ampicillin (Amp)?

Remember that bacteria that do not pick up plasmid cannot grow and therefore your are selecting for only those that have been transformed.Summary: Two selection criteria:

1. Ampicillin resistance showing presence of plasmid

2. White colony showing presence of insert.

Fig. 12.3


Review Slide


Review Slide

What is the problem with this if we were subcloning a eukaryotic gene?INTRONS!! If you take a eukaryotic gene and insert it straight into a vector, the introns are still there and bacteria cannot splice out introns.

How do we fix this eukaryotic gene problem?

Why can’t they splice out introns?Because they do not have

introns!

Fig. 12.7

Let the eukaryotic cell take out the introns for you…Instead of taking the gene from the eukaryotic cell, take the processed mRNA.

But this leads to another problem, we can’t put RNA into a DNA plasmid…


Fig. 12.7

Make cDNA (complementary DNA) from the mRNA:

Advantages to cDNA 1. No introns2. No junk DNA

1. Isolate mRNA

2. use reverse transcriptase to make a dsDNA copy3. cut with restriction enzyme and ligate into a vector


Summary1. Isolate plasmid


2. Isolate gene of interest (straight from genome if bacterial or via mRNA if eukaryotic)3. Cut both with same restriction enzyme 4. Mix together to allow sticky ends to ANNEAL forming recombinant DNA5. Ligate using DNA ligase

6. Transform bacteria with vector (plasmid)7. Bacteria will express (make) the protein and divide making more copies of the gene (gene cloning)

Conclusion

We can make any protein we want or more of any gene (gene cloning) by putting it into a plasmid and transforming a bacterium.


There is another, more efficient way of making more of any gene or DNA segment we want…using a method called:

Chapter 20 - DNA Technology and GenomicsAIM: What are some other tools of DNA technology?

PCR (Polymerase Chain Reaction)Technique used to amplify (make more of) a

specific piece of DNA. Can be a gene or any other segment. It is essentially DNA replication in a test tube with a twist…

http://www.maxanim.com/genetics/PCR/PCR.htm


http://www.youtube.com/watch?v=x5yPkxCLads

PCR


1. Denaturing2. Annealing 3. Extending

Combine template DNA, DNA primers flanking the target region, Taq polymerase, deoxynucloside triphates

A crime has been committed and you have a suspect as well as a tiny bit of DNA sample from the scene of the crime. What do you do?


The first thing you do is PCR the DNA to make more copies of it…

Let’s assume you go ahead and do this…now what?

Fig. 12.11A

**Everyone’s DNA has a slightly different sequence (every 1 in 1000 bases is different), so we all have different restriction site patterns.


Amplified section of the DNA from the crime scene

The PCR amplified portion of this person has two restriction sites.

How many restriction fragments (DNA pieces) would there be after cutting with the restriction enzyme?

three

You have a suspect. What should you do?


Use PCR to amplify the same segment of the subjects DNA and cut it with the same restriction enzyme.

Fig. 12.11A

twoHow many restriction fragments will the suspects DNA yield?



Amplified section of the same DNA segment from the suspect.

The suspect has a different allele with a mutation in the first restriction site. The restriction enzyme will not cut this sequence.

The suspect did not commit the crime.

Conclusion:

Crime scene DNA Suspect DNA

Restriction fragment length polymorphisms (RFLP’s = “rif lips”)




The differences in restriction sites found on homologous chromosomes giving rise to different numbers and lengths of restiriction fragments.

Crime scene DNA Suspect DNA

Fig. 12.11A




This is great, but you can’t see DNA restriction fragments directly so how will we actually count the fragments?How can we OBSERVE the DNA restriction fragments?

Fig. 12.10

Gel Electrophoresis

This technique allows one to separate DNA fragments by size and view the DNA fragments.


cathode

anode

Fig. 12.10

Gel Electrophoresis


http://www.youtube.com/watch?v=QEG8dz7cbnY

Gel Electrophoresis


As you saw in the video, the researcher put a chemical called ethidium bromide (shown below)into the gel solution.

This compound binds to DNA (right, below) and fluoresces when hit with UV light thus allowing us to see where the DNA is located in the gel (right, above).

Fig. 12.10

Gel Electrophoresis


Gel (like jell-o)

The gel is made of either agarose or polyacrylamide. It has tiny, microscopic pores that DNA can fit through.

cathode

anode

Fig. 12.10

Gel Electrophoresis


The DNA sample is loaded in the wells at the top of the gel. One sample per well.

Gel (like jell-o)

cathode

anode

Fig. 12.10

Gel Electrophoresis


Electricity is then run through the gel. Why do you think the negative end is on the sample side and the positive end is on the other end of the gel?

Electricity (electrons flow from top of gel by the samples to the bottom of the gel)

DNA is negative because the phosphates are negative. The negative electrons moving down push (repel) the DNA down with them.

cathode

anode

Gel Electrophoresis


Which will move faster through the micro-porous gel, the longer DNA fragments or the shorter DNA fragments?The small fragments (fewer nucleotides) will move more easily through the gel and hence go faster than the large ones. Therefore, gel electrophoresis separates DNA fragments by SIZE.

cathode

anode

Fig. 12.10

Gel Electrophoresis


This is all great, but we still can’t physically see the DNA…

cathode

anode

Fig. 12.10

Gel Electrophoresis


The gel is soaked with a a compound called ethidium bromide, which sticks to DNA and lights up when you hit the gel with UV light…

cathode

anode

Gel Electrophoresis


http://www.youtube.com/watch?v=Wwgs-FjvWlw&feature=related

You are not observing the DNA move. You are seeing a blue dye added to the sample move through the gel. You cannot see the DNA until you put the gel under a UV lamp as discussed before.

AIM: What are some of the other tools of DNA technology?

Virtual Lab

(http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/virgel.html)

http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/virgel.html


Draw what the gel would look like for the restriction digest of the criminal and the suspect.



Fig. 12.11Acriminal suspect

Suspect’s DNA fingerprint

Criminal’sDNA fingerprint


Can also be used to determine:

1. Paternity (allele 1 from child, allele 2 amplified from suspected father).


Can also be used to determine:

2. Diseases resulting from DNA changes that alter restriction sites.


Review:


1. Use PCR to get more of the desired DNA2. Digest DNA with restriction enzymes3. Run restriction fragments on a gel (gel electrophoresis)4. Compare fragments

Question: You have been given two DNA samples that have gone through PCR. Both samples are of the same DNA segment with a size of 1kb (1 kilobase = 1000bp). Sample 1 has four restriction sites at 100bp, 300bp, 350bp, and 700bp. The second piece has the same sites in addition to a fifth site at 725bp. Draw how the gel should look for these two pieces.



Sample 1

Sample 2

100bp 300bp350bp 700bp

Segments of DNA:

50bp, 100bp, 200bp, 300bp, 350bp

100bp 300bp350bp 700bp

725bp

Five segments in total -

25bp, 50bp, 100bp, 200bp, 275bp, 350bp Six segments in total -


-

+

Do not forget to label the charges on the gel and show the flow of electrons (the current).

50bp

100bp

200bp

300bp

350bp

50bp

100bp

200bp

350bp

25bp

275bp

Sample1

Sample2

e-

Gel electrophoresis can be done using proteins as well. In this case the gel is made of polyacrylamide and the proteins are coated with negatively charged molecules called SDS since they are not always negative like DNA. It is a little more complicated, but not much…


“Pharm” animalsFig. 12.16


NEW AIM: Making transgenic organisms.

Fig. 12.18AB


AIM: Making transgenic organisms.

Transforming plants:

by electroporation

An infectious soil bacterium, Agrobacterium tumefaciens, contains a plasmid known as Ti plasmid that naturally integrates a section of its plasmid into the plant’s DNA.

We have isolated the plasmid, rendered it non-infectious, and put desired genes into the part of the plasmid that gets incorporated known as the T DNA.



Transgenic mice have been invaluable tools:We have the ability to add or take away any gene we want from mice to observe the affect of that gene.Two methods are available to do this:1. Transform embryonic stem cells2. Inject desired gene into male nulceus after fertilization, but before fusion of nuclei occurs



An example:Normal mice cannot be infected with polio virus. They lack the cell-surface molecule that, in humans, serves as the receptor for the virus. So normal mice cannot serve as an inexpensive, easily-manipulated model for studying the disease. However, transgenic mice expressing the human gene for the polio virus receptor

* can be infected by polio virus and even * develop paralysis and other pathological changes characteristic of the disease in humans.

Transgenic mice have been invaluable tools:

Fig. 12.19

Chapter 20 - DNA Technology and GenomicsAIM: Making transgenic organisms.

Gene Therapy

- Replacing a defective gene with a normal gene.

chapter 20 - dna technology and genomics genetically modify organisms and transgenic organisms...

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