1. Feeding a growing world pGLO transformation of E. coli

2. Transformation is… the genetic alteration of a cell… resulting from the uptake of exogenous DNA (DNA from outside the cell)… through the cell membranes… so that the foreign DNA is incorporated into the transformed cell.

3. Transformation is… Transformation occurs naturally in some species of bacteria. The bacteria must be in a state of competence. Competence is the ability to take up exogenous DNA. This state of competence might be a response to population density or starvation.

4. History In 1928 Frederick Griffith, a British bacteriologist, discovered that a non-virulent strain of Streptococcus pneumoniae could be made virulent by exposing it to heat-killed virulent strains. He could not explain the results.

5. History In 1944 Avery, Macleod and McCarty recognised that this transformation was due to a genetic change. They isolated DNA from a virulent strain of S. pneumoniae, injected it into a harmless strain and made the harmless strain virulent. This group coined the term ‘transformation’.

6. History Their results were greeted with scepticism… until 1953, when other research demonstrated that bacteria can take up DNA by conjugation (horizontal transfer) and transduction (introduction of phage virus DNA).

7. History It was originally thought that E. coli bacteria could not be transformed, but in 1970 Mandel and Higa showed that E. coli could take up DNA, without a helper phage virus, after treatment with calcium chloride solution. This method was improved by Douglas Hanahan and is the basis for the colony transformation used in the experimental protocol you are about to use.

8. Uses Genetically transformed bacteria produce a protein encoded by the transferred gene, so the technique of genetic transformation has many uses in biotechnology: In agriculture – GM crop plants that yield more or resist drought, disease or pesticides In medicine – GM E. coli that produce insulin In bioremediation of oil spills – GM bacteria that digest toxic components in oil.

9. In some countries a decorative GM GloFish TM is commercially available. It was modified with a gene for green fluorescent protein. Uses Photo: glofish.com

10. Practical investigation In the previous activity you learned about the potentially harmful effect of high reducing sugar content in potatoes, along with how potatoes might be modified to have a lower sugar content. The techniques you are about to try are used by scientists developing low-reducing-sugar potato varieties to decrease the amount of acrylamide in potato products.

11. Practical investigation In this practical investigation, you will use the techniques of genetic modification to insert a gene from one species into another (the bacterium E. coli). The insertion is carried out using a plasmid, a ring of bacterial DNA, as a vector to carry the gene into the recipient. The inclusion of a gene for green fluorescent protein enables you to see immediately whether a cell has taken up the DNA from the plasmid.

12. The gfp gene is expressed in the eyes of these fruit flies (Drosophila melanogaster). Practical investigation Photo: Derric Nimmo & Paul Eggleston/Wellcome Images

13. In this protocol you will transform E. coli with a gfp gene from the jellyfish Aequorea victoria. Practical investigation The crystal jelly, Aequorea victoria, a bioluminescent jellyfish from the Pacific coast of North America. Photo: Denise Allen

14. The transformed bacteria will express the gfp gene and produce green fluorescent protein (GFP). Practical investigation pGLO TM -transformed E. coli exposed to ampicillin and ultraviolet light. Photo: Thenickman100

15. The pGLO TM plasmid oriorigin (point at which replication starts) araCarabinose promoter gfpgene coding for GFP blaampicillin resistance gene

16. The pGLO TM plasmid Recognition sites for the restriction endonuclease enzymes NdeI, EcoRI and HindIII are present. Once the plasmid is cut it can take up the gfp gene. The gene araC encodes a protein: a transcription factor. When this factor binds to arabinose, transcription of the gfp gene can proceed.

17. The controls –pGLO bacteria plated onto agar medium with neither arabinose nor ampicillin: demonstrates that the bacteria are viable after exposure to calcium chloride and heat-shock treatment. –pGLO bacteria plated onto ampicillin agar: do not have the ampicillin resistance gene as they have not been transformed. They should not grow. If they do, they have undergone a mutation and are no longer suitable for use in this protocol.

18. +pGLO on amp agar This shows that the transcriptional control of the gfp gene is intact as no GFP is made in the absence of arabinose.

19. +pGLO on amp/ara agar This is the experimental plate. These bacteria are transformed and are the only ones that will produce GFP. They have taken up the plasmid, so they have the gfp gene, and arabinose is available to switch on expression of this gene. The bacteria will glow green under long- wavelength UV light.

20. Links to video Bacterial transformation using pGLO TM plasmid as a vector (Bio-Rad) http://thecrunch.wellcome.ac.uk/schools Making fish oils in plants: use of GM technology to replace a diminishing natural resource (Prof J Napier, Rothamsted Research) http://thecrunch.wellcome.ac.uk/schools