In May, students in the Biology Department produced WHS’ first

Genetically Modified Organism...

More detailed science

Experimental results

Overview

Photo gallery

Bacterial transformation

was carried out using a gene

from the bioluminescent

jellyfish

Aequorea victoria

Experimental Overview

Gene for green fluorescent protein (from the jellyfish)

Gene for beta-lactamase (confers antibiotic resistance)

The transformation was carried out using a pGLO plasmid which had been modified to contain two new genes..

If we could get pGLO into the bacteria, they would be antibiotic resistant and would emit

green light under a UV lamp

Safety

Escherichia coli strain HB101 K-12 does not produce compounds or toxins that can make people sick.

Escherichia coli strain HB101 K-12 cannot survive outside the laboratory.

Basic Principles

Students were working with tiny quantities (100μl); They could hardly see any liquid at all!

Quantities and timings were vital. The heat shock (50s at 42 o C) had to be timed precisely and transfer to ice needed to be immediate.

More detailed science...

Or jump to:

Experimental results Photo gallery

More detailed science...

If the pGLO plasmid is taken up, the genes for ampicillin resistance and GFP will be present.

Bla is the beta-lactamase gene.

Inside bacterial cells it is transcribed into mRNA, then translated to produce the beta-lactamase enzyme.

This enzyme inactivates ampicillin (an antibiotic).

Why is bla important in this experiment?

araC is a gene required to promote the transcription of the arabinose operon (a series of genes that digest arabinose sugar).

araC needs to bind with arabinose sugar in order to function as a promotor.

If arabinose is absent, the gene is ‘silent’.

What is the advantage of this to the bacterium?

Normally the operon has 3 genes coding for enzymes that break down arabinose sugar

The gene is only expressed (transcribed) in the presence of arabinose

araC needs to bind with arabinose before it allows pBAD to promote transcription.

Arabinose is a food source for bacteria

In the pGLO plasmid, the arabinose operon (araA, araB and araD genes) have been replaced with the gene for GFP.

If arabinose is present, it will promote transcription into mRNA, which will be translated into GFP.

RESULTS – MAY 2014

These E. coli bacteria have been modified with the pGLO plasmid.

They are now resistant to the antibiotic ampicillin which was added to the growth medium.

(Megan & Connie, Year 11)

These E. coli bacteria have also been modified with the pGLO plasmid and are resistant to the antibiotic ampicillin.

These colonies have been grown on nutrient agar containing arabinose sugar, which switches on the gene expression for GFP (Green Fluorescent Protein).

(James, Year 13)

Under UV light, the modified bacteria express GFP only when grown on nutrient agar containing arabinose sugar.

Our aseptic technique was not good enough to prevent contamination, but we did manage to transform two bacteria.

(Tom & Jasper, Year 10)

Photo gallery

This practical work was made possible by the school’s More Able & Talented programme, and the superb support provided by the biology technicians.

Thank you!