Biotechnology & SynBioBy C. Kohn, Waterford WI

Better BushelsImagine if we had a crop that produced

its own pesticide…◦We wouldn’t have to spray fields with a

chemical pesticide◦This would save farmers money and protect

the environment◦ It would also reduce the pathogens that

affect our crops, reducing the need for other protective treatments while making fields more productive.

Would this be a good thing or a bad thing? Discuss

Bt CornSuch a product does exist and has been

used for over 15 years.

In fact, 45% of the corn grown in the US is genetically modified.

The most famous example of this is Bt Corn. ◦Left: Control◦Right: Bt Corn

Bt CornBt Corn is a GMO, or Genetically Modified

Organism. ◦GMO: a plant or animal that has been genetically

modified through the addition of a small amount of genetic material from other organisms.

In a GMO, genetic material from another organism is inserted into the plant or animal genome.

These genes can come from any living source, including bacteria, fungi, and other organisms.

Bt = Bacillus thuringiensis In the case of Bt Corn, an inserted gene for a

natural insecticide came from Bacillus thuringiensis, a bacterium found naturally in the soil. ◦ B. thuringiensis bacteria naturally produce a toxin (the

Bt delta toxin) which kills specific predatory insects during the larval stage.

◦ It does not harm other insects in the way broad-spectrum insecticides do, making it an ideal replacement to synthetic chemical pesticides. Bt was actually available

as a separate pesticide since 1960 and has an excellent safety record, making it an ideal choice as a GMO.

Production of Bt CornProduction of Bt Corn was relatively

straightforward: 1. The gene for the Bt toxin was sequenced and

identified. 2. The gene was removed from the B. thuringiensis

genome using a restriction enzyme. 3. The genome of corn was spliced using the same

restriction enzyme. 4. The gene was inserted and made permanent

using DNA ligase. 5. The modified corn genome was inserted into a

corn cell nucleus. 6. The corn cell, when it divided, produced the Bt

gene along with the rest of the corn’s genome.

Bt in action.Because Bt corn has the gene for the Bt toxin, it

produces this protein just like any other protein in a corn cell.

When an insect ingests the Bt toxin protein produced by the corn, the Bt toxin binds to the stomach wall of the insect.

Within hours the stomach wall is broken down by the toxin.

Bt & Monarchs Concern has been raised about the

impact of Bt corn on monarch butterflies.

Research by the USDA’s Agricultural Research Service has shown that other than an early version of Bt Corn (which has since been replaced), the impact on monarchs is negligible and insignificant.◦ Plus, the alternative to Bt corn is the use

of chemical pesticides, which are far more harmful to butterflies.

Is It Safe? Bt corn was approved by the USDA for human consumption in

1995. Is it safe?◦ This might be a good question, given the Bt toxin kills insects by

destroying their intestinal tracts

“Delta endotoxins and VIPs produced by the currently available events all are rapidly broken down in the stomach and thus are not potential food allergens.” – Colorado State University ◦ i.e. your own stomach will rapidly break down the toxins before they

can affect you

Bt corn is considered generally safe a not a threat to consumers. ◦ It is regulated by both the

EPA and FDA for human and environmental safety.

◦ It has been used for over 15 years with no record of serious issue.

Biotechnology Bt Corn has become the poster child of biotechnologies made

possible by recombinant DNA.

Recombinant DNA has made the science of biotechnology possible. ◦ Recombinant DNA: when genes from two different species are

combined and introduced into a cell◦ Biotechnology: the manipulation of the genetics of organisms to

make useful products

Biotechnology is not necessarily a new science◦ Selective breeding of livestock and the use of microbes to make

wine are ancient examples of biotechnology

However, with the use of recombinant DNA and other technologies, biotechnology has changed modern life.

Making Recombinant DNAProduction of recombinant DNA is similar

regardless of what you are producing.

First, a gene must be cut using a restriction enzyme (a chemical scissors for DNA that always cuts at the same sequence of bases)◦ Copies of DNA always

yield the same restriction fragments when exposed to a restriction enzyme (meaning DNA copies are always cut in a predictable way).

Making Recombinant DNAIf a restriction enzyme cuts DNA in such a

way that a single-stranded portion remains, this is called a “sticky end”

Sticky ends are important because they allow the addition of new genes so long as they have the complementary sequence to the sticky ends◦ E.g. a new gene would have

to have a TTAA sticky end to ‘fit’ inside these restriction fragments.

Creation of Recombinant DNA1. A restriction enzyme cuts DNA2. Restriction fragments are created3. A new gene with complementary

sticky ends is inserted. 4. DNA ligase (an enzyme) permanently

seals the new gene into the genome.

RestrictionEnzymeDNALigase

DNA LigaseDNA ligase enzyme is necessary to “cement”

the new gene into the genome.◦ Without DNA ligase, the bond is only temporary. ◦ DNA Ligase is the “super glue” that makes a

bond permanent

Once DNA ligase has formed a permanent bond with the new gene and the original genome (the “vector), we have recombinant DNA. ◦ A cloning vector is the

DNA that carries the inserted gene

DNALigase

Biofuels Scientists are currently working to develop GM

organisms that can convert cellulose (in plant cell walls) into (such as ethanol and biodiesel).

Scientists are working to identify the genes responsible for more efficient breakdown of cellulose and how and where to insert these genes into the genome of modified organisms like E. coli and yeast.

The hope is that an organism could be developed to either breakdown cellulose more efficiently than we currently can with yeast produced through selective breeding.

Synthetic Biology The use of recombinant DNA in biotechnology has led to

the rise of a new science: synthetic biology.◦ Synthetic Biology: a science combining engineering,

biotechnology, and biology in order to create new biological organisms that do not exist naturally in the environment.

In a sense, synthetic biology is the combination of genes from different sources to create a “super organism” that can do all the things we need it to do.

By selectively choosing and inserting genes, the hope is that we can create organisms from scratch that can address the most pressing needs facing society.

Examples of Syn-Bio AttemptsSynthetic Biology has been used so far to

address the following: ◦ The production of more productive microbes for

the rumens of cattle◦ Production of more potent and effective vaccines◦ Production of bio-engineered drugs that target

and destroy cancer tumors◦ Bioremediation microbes that can quickly clean

up an oil spill or toxic waste ◦ Biofuel-producing microbes that can convert

plant feedstocks into more useful substances (cellulose into glucose for ethanol or oil for biodiesel).

The Moral and Ethical ImplicationsIf a product of synthetic biology were to

escape into earth’s fragile, complex and highly interdependent ecosystem, it could have devastating effects on all naturally occurring organisms. ◦ Our “super organisms” could become super-

invasive, seriously disrupting natural processes.The release of synthetically engineered

organisms may have damaging side effects. Many who oppose this type of research fear

that this science could be abused may fall into the hands of terrorist groups and rogue nations.

Current ResearchWatch the following video

Recombinant DNA, as advanced as it is, is even being phased out by advances in SynBio.

Use of recombinant DNA was sort of like writing a book using words and sentences from other books.◦ Scientists now are trying to create genes from

scratch that could be inserted into cells, enabling them to write their “book” from scratch rather than trying to find the genes responsible for the traits they are trying to create. .

SynBio & MalariaOne of the first major successes with Synthetic

Biology was the production of precursor to the compound artemisinin.◦ This compound has been shown to be effective

against strains of malaria that are resistant to more widely-used drugs

◦ However, this drug was far too expensive to produce

"By inserting genes from three separate organisms into the E. coli, we're creating a bacterial strain that can produce the artemisinin precursor." Jay Keasling, lead researcher on the project.

SynBio Malaria MedicineKeasling and his team took genes

from yeast and from the sweet wormwood tree (the source of the drug compound) and inserted them into E. coli. ◦As a result, the yield of the artemisinin

precursor in that strain of E. coli was increased by 10,000 times compared tothat of the wormwood tree. Right: Keasling and the

wormwood tree Photo courtesy of: www.lbl.gov

Biofuel of the Future If we were to create a similar organism as

the malaria-drug E. coli bacterium, what traits would it need?

Think/Pair/Share – what kinds of genes would we need to insert into yeast or E. coli in order to produce the ideal biofuel microbe?

What aspects of this work might interfere with the function of this modified organism? ◦E.g. does it matter where the gene is placed?

Problems With SynBio If we continue our book analogy, writing a book is far

harder than copying and pasting text from other publications (which is why plagiarism is so bad among students;)

For example: does the placement of the word in a sentence matter?◦ In sentence: does the matter of the example in a of matter

placement?◦ Obviously, it does.

The same is very true for genes – we can’t just lob it in the genome somewhere – the placement of a gene will very much affect how effectively the gene is expressed.

More ProblemsAnother problem with SynBio, as well as any

modified genetics, is that modified organisms can lose their competitiveness as they become more and more modified.

Many modified organisms lack the ability to compete to the extent that they cannot function outside of a lab setting.◦ Obviously this would be a problem for the industry.

There is no standard way to modify an organism and make it competitive and functional outside of a lab. ◦ This makes trial and error a big part of this process.