GM bacteria

Question 1

What are the applications of Genetic engineering?

Answer 1

Research- transgenic bacteria to produce DNA and proteins

Agriculture- Corn, Hawaiian papaya, soybeans, cotton, sugar beets, canola, alfalfa

Medicine- synthetic insulin, human growth, hormones, vaccines, CAR-T

Question 2

When was the first genetically engineered synthetic human insulin produced?

Answer 2

1978
First genetically engineered synthetic human insulin produced in E. coli at Genentech
Humulin brought to market in 1982

Question 3

What can be used as a factory to produce proteins?

Answer 3

E. coli

Question 4

What are used as cell factories?

Answer 4

Genetically modified microbes

Question 5

How do we produce drugs such as insulin?

Answer 5

1. Clone human insulin gene into plasmid
2. Transform bacteria with plasmid
3. Select transformed bacteria
4. Culture bacteria and induce gene expression
5. Lyse cells
6. Purify protein with chromatography
7. Analyze purification with SDS-page
8. Assay protein activity

Question 6

How was insulin genetically engineered?

Answer 6

The gene that codes for human insulin was initially genetic engineered into a plasmid and expressed in bacteria in 1978 by scientists at Genentech. This was the first ever recombinant protein developed for a pharmaceutical application

Question 7

What does a generic plasmid look like?

Answer 7

Ori- origin of replication allows bacteria to make copies of plasmid via DNA polymerase

Gene- gene of interest cloned into plasmid

Question 8

How is a gene cloned?

Answer 8

1. Isolate gene of interest and cut with restriction enzymes (or use PCR)
2. Cut with plasmid with same restriction enzymes
3. Insert gene into plasmid using DNA ligase

Question 9

How does bacterial transformation take place?

Answer 9

Genetic transformation occurs when a cell takes up DNA and expresses the genes on that DNA
Heat shock
Electroporation
The bacteria divide and the new bacteria also have plasmids
Genes are transcribed and translated

Question 10

How do we know the E. coli is transformed?

Answer 10

Antibiotic selection
Marker genes

Question 11

What is GFP?

Answer 11

Green Fluorescent Protein (GFP)
GFP allows us to visualize protein expression with UV light

Question 12

How can GFP be utilised?

Answer 12

GFP as a visual tracer/marker
Study of biological processes (such as protein synthesis)
Localisation and regulation of gene expression
Cell movement
Cell fate during development
Formation of different organs
Screenable marker to identify transgenic organisms
Cellular localization of drugs

Question 13

What is an example of GFP being used?

Answer 13

GloFish
Aquarium lovers have used genetically engineered fish with GFP
Is a concern that transgenic animals like these accidentally becoming established in nature

Question 14

What does a plasmid with the gene for GFP look like?

Answer 14

GFP can be used as a marker gene
Ori- origin of replication allows bacteria to make copies of plasmid

GFP gene- encodes GFP

Question 15

Can plasmids carry multiple genes

Answer 15

Yes

Question 16

What does the plasmid for coding for beta-lactamase (amp) gene look like?

Answer 16

Beta-lactamase (amp) gene for antibiotics resistance
Ori- origin of replication allows bacteria to make copies of plasmid

amp (or bla) gene- encodes beta-lactamase an enzyme that breaks down ampicillin (an antibiotic)

GFP gene- encodes Green Fluorescent (GFP)

Question 17

How is the gene for Beta lactamase (amp) produced?

Answer 17

Transformation with plasmid with amp
Bacteria plated on ampicillin plates
Beta lactamase produced
Beta lactamase breaks down ampicillin
Bacteria that aren’t transformed don’t grow
Transformed bacteria grow into colonies

Question 18

What does beta-lactamase do?

Answer 18

Beta lactamase provides antibiotic resistance by breaking down the antibiotic
They cleave the beta-lactam ring of some medically important beta-lactam antibiotics such as ampicillin

Question 19

What does a pGLO plasmid look like when expressing GFP?

Answer 19

Ori- origin of replication allows bacteria to make copies of plasmid

araC gene- encodes the AraC repressor protein

pBAD promoter- landing site for RNA polymerase to transcribe the downstream gene

amp (or bla) gene- encodes beta-lactamase, an enzyme that breaks down ampicillin

GFP gene- encodes Green Flurorescent Protein (GFP)

Question 20

What is arabinose and how does it relate to AraC?

Answer 20

AraC acts as a repressor in the absence of arabinose
Arabinose (inducer)

When arabinose binds AraC, the AraC comformation changes, activating the expression of the araBAD genes that metabolize arabinose

Question 21

What happens without arabinose?

Answer 21

Without arabinose, AraC acts as a repressor- a loop is formed in the DNA, preventing RNA polymerase from transcribing araBAD genes

Question 22

What happens with arabinose?

Answer 22

With arabinose, AraC undergoes a conformational change, allowing RNA pol to transcribe the araBAD genes

Question 23

For GFP expression, what happens with arabinose?

Answer 23

Without arabinose, RNA polymerase is blocked from transcribing the GFP gene
However, with arabinose, RNA polymerase can transcribe the GFP gene