PLANTS AS BIOREACTORS

Bioreactors
- A system designed to obtain an effective environment for the conversion of one material into some product by
appropriate biochemical reactions

How can plants be bioreactors?

• Plants are usually genetically modified in order to produce certain substances such as hormones. Genes from
different sources (e.g. humans, bacteria, animals) are integrated into the plant’s genome.

Reasons of Using Plants as Bioreactors

• There is stable integration of foreign DNA into plant genome
• Processing and assembly of foreign proteins in plants are similar to those in animal cells
• Plants are inexpensive to grow in a large scale
• Not limited by fermentation capability
• Foreign genes might be produced in seeds and will be maintained for long periods under ambient conditions

Proteins Naturally Produced by Plants

• Monellin and thaumatin – sweetest-known naturally occurring substances
- Produced by serendipity berries and katemfe

• β-amylase- During the ripening of fruit, β-amylase breaks starch into maltose, resulting in the sweet flavor or a
ripe fruit

• Papain- a proteolytic enzyme extracted from the raw fruit of the papaya plant. Proteolytic enzymes help break
proteins down into smaller protein fragments called peptides and amino acids. This enzyme is frequently used in
tenderizing meat.

Foreign Proteins Produced in Plants

Protein Source Host plant
Alpha amylase bacteria tobacco
Erythropoietin human tobacco
Glucoamylase fungi potato
Growth hormone trout Tobacco
Hepatitis B surface Virus Tobacco

• Human growth hormone -The first foreign protein produced from tobacco
• Human glucocerebrosidase - Most expensive drug; breaks down glucocerebroside

*** “Plant Vaccines” - Formerly called as “Edible Vaccines”

*** Beta-carotene – precursor of Vitamin A; found in ‘Golden Rice’
*** Lycopene – a powerful antioxidant found in tomatoes

Advantages
• Likely economically attractive production cost
• Ease of scale up
• Availability of established practices equipment for plant harvesting/storage
• Ability to carry out post-translational modification
• Plant pathogens do not infect animals or humans

Disadvantages
• Low expression levels often reported
• Lack of industrial experience or data on large scale downstream processing of plant tissue
• Seasonal or geographical nature of plant growth
• Availability of established alternative production systems
 BIOTERRORISM

Bioterrorism - A type of warfare that makes use of biological agents to inflict harm to the enemy

What are used as biological weapons?

• Pathogenic (disease-causing) bacteria and viruses are often used as biological weapons. Substances such as
toxins and antigens produced by microorganisms, along with poisonous and toxic substances extracted from
plants can also be used in bioterrorism.

Categories of Biological Weapons

1. Category A
• high-priority agents that pose a risk to national security
• can be easily transmitted and disseminated
• result in high mortality
• have potential major public health impact

• examples of Category A biological weapons:

1. Anthrax - caused by Bacillus anthracis, a bacterium that produce spores that makes toxins
- can be treated with antibiotics
- types of anthrax:
a. Cutaneous Anthrax – results when anthrax enters an open wound
b. Intestinal Anthrax – caused by the ingestion of meat contaminated with
anthrax
c. Inhalation Anthrax

2. Botulism – caused by the bacterium Clostridium botulinum which releases a toxin that can
paralyze the muscles
- Obtained from improperly canned foods or fish
- causes abdominal pains, nausea, vomiting, diarrhea

3. Plague - also known as Black Death or Black Plague

- caused by Yersinia pestis, a bacterium found in rats and are transferred to humans
through lice and ticks

4. Smallpox - caused by the virus Variola

2. Category B
• moderately easy to disseminate and have low mortality rates
• examples:
1. Glanders – caused by the bacteria Burkholderia mallei
- Once people are infected, they develop a fever and rigors. Eventually, they get pneumonia, pustules,
and abscesses, which prove fatal within a week to 10 days if left untreated by antibiotics.

2. Ricin - A poison derived from castor bean plants

- Ingestion can cause intestinal bleeding and organ damage

3. Typhus - Common symptoms include fever, headache, and a rash. Typically these begin one to two weeks after
exposure.

3. Category C
• Category C agents are emerging pathogens that might be engineered for mass dissemination because of their
availability, ease of production and dissemination, high mortality rate, or ability to cause a major health impact.
• Examples: H1N1 virus (Swine flu virus)
HIV / AIDS
Why do people use biological weapons?
Biological weapons are:
• Invisible or microscopic
• Simple laboratory techniques are required for preparation of these agents and may not require sophisticated
apparatus
• Easy to multiply and maintain
• Difficult to trace
• Very deadly

DNA FINGERPRINTING

• DNA fingerprinting is the determination of an individual’s unique collection of DNA restriction fragments

Why use DNA Fingerprinting?

• DNA fingerprinting is a way of telling individuals of the same species apart
• DNA sequences are variable and can therefore be used as identifying characteristics.
• Why use DNA Fingerprinting?
• DNA fingerprinting has advantages over other sources of evidence (fingerprints, blood type, etc.):
– Highly accurate.
– Can be gathered from trace crime scene evidence.

Steps in DNA Fingerprinting

1. Collecting DNA sample
• Extract DNA from a sample of human material
• Examples: blood, saliva, hair, semen

2. Extracting DNA sample from its source

• The extraction process is devised in a way to break down the cell membrane and release the DNA to its outer
environment.

3. DNA Replication
• The DNA sample extracted from a source is needed to be replicated in order to have more DNA strands to
analyze.
 Polymerase chain reaction (PCR)
• A lab technique used to amplify segments of DNA

4. Restriction Enzymes
• Once the DNA is extracted, it is subjected to digestion by restriction endonucleases. These are enzymes that cut a
DNA fragment at specific sites which it recognizes. It means that each restriction enzyme (R.E.) recognizes a
specific DNA sequence and cuts at a specific site.
• Restricted Fragment Length Polymorphism (RFLP)
• Nucleotide sequence variations in a region of DNA that generates fragment length differences according
to the presence or absence of restriction enzyme recognition sites.
• Variable Number Tandem Repeat (VNTR)
• sequences that are repeated multiple times and the number of repeats varies from person to person

5. Gel Electrophoresis Technique

• The DNA was loaded into wells at one end of a porous gel, which acted a bit like a sieve.
• An electric current was applied which pulled the negatively-charged DNA through the gel.
• The shorter fragments move faster, thus at the end of this process, they are closer to the positive end of the
agarose gel

6. Denaturation of DNA Strands

• The gel containing DNA fragments are then immersed in an alkaline environment.
 • This step helps in denaturing DNA strands into single stranded DNA.

7. Southern Blot Technique

• Transferring single stranded DNA fragments on to a nylon membrane.

8. Probing/Hybridization
• Adding radioactive or colored probes to the nylon sheet produces a pattern called the DNA fingerprint.
• This DNA probe is a single stranded DNA having a sequence complementary to the desired sequence
• Under X-ray, the probes attached to the DNA fragments will “light up”