1. Key Area : Genetic Control of Metabolism in Micro-organisms Unit 2: Metabolism and Survival

2. Selection and isolation Wild strains of microorganisms can be selected to be of use in industrial processes. What is meant by wild strains? These can then be cultured in an enriched nutrient media and pure strains of the microorganism isolated. CfE Higher Biology Metabolism and Survival

3. Selection and isolation Pure strains still may need to be improved. – To improve genetic stability – To improve their ability to grow on low cost nutrients – To produce large quantities of target compound – To allow easy harvesting of target compound after fermentation is complete These improvements can be brought about by mutagenesis, breeding programmes or recombinant DNA technology. CfE Higher Biology Metabolism and Survival

4. Altering microorganisms Give a brief description of how mutagenesis, selective breeding programmes and recombinant DNA technology all produce altered microorganisms. CfE Higher Biology Metabolism and Survival

5. Mutagenesis Mutagenesis is the process of inducing mutants. In nature, they are rare, spontaneous and cause variation. Rate of mutagenesis can be increased by exposure to mutagenic agents. Name some mutagenic agents. UV light, forms of radiation (X-rays, gamma rays), chemicals such as mustard gas. CfE Higher Biology Metabolism and Survival

6. Mutagenesis Improved characteristics can be shown by mutant strains, such as increased yield. These strains can be genetically unstable and reverse mutation can occur e.g. a deletion in DNA can be repaired by the subsequent addition of DNA. CfE Higher Biology Metabolism and Survival

7. Mutagenesis The useful mutated microorganism can then be used in industry. However it must be monitored closely if used in industrial fermenters because if a less useful type is used this will impact on the materials used and produced in the process and the time taken for the process. This all impacts on the costs involved. CfE Higher Biology Metabolism and Survival

8. Breeding programmes Bacteria reproduce asexually and therefore variation is not introduced through reproduction. New strains of bacterial species can arise due to horizontal transfer of genetic material. Plasmids or chromosomal DNA can be transferred this way. DNA fragments may also be taken up by bacteria and incorporated into their DNA from their surroundings producing new strains. CfE Higher Biology Metabolism and Survival

9. Recombinant DNA technology This allows scientists to transfer gene sequences from one organism to another and from species to species. A protein may therefore be produced from an artificially transformed microorganism. What other improvements can be made to a microorganism by introducing genes? CfE Higher Biology Metabolism and Survival

10. Recombinant DNA technology Improvements such as: Amplifying specific metabolic steps in a pathway which will cause a increase in the yield of a target compound. Cause cell to secrete product into surrounding medium as this will make it easier to harvest. Prevent microorganism from surviving in external environment which reduces chance of outbreak of microorganism. CfE Higher Biology Metabolism and Survival

11. Artificial transformation of a bacterium Genes for desirable characteristics can be selected by scientists. A well known example is the insulin gene. The gene of interest is removed from its normal position and spliced into the DNA of a vector. Vectors are usually bacteria, e.g. E.coli When the bacteria is cultured it produces the product formed by the inserted DNA. This would be the hormone insulin in the case of the insulin gene. CfE Higher Biology Metabolism and Survival

12. Artificial transformation of a bacterium Write a description and an explanation on the use of recombinant DNA technology using the information and diagram on page 208 of your text book. CfE Higher Biology Metabolism and Survival

13. Recombinant DNA tools A restriction endonuclease is an enzyme extracted from bacteria. It is used to cut up DNA into fragments. The fragments usually contain specific genes that are required. These enzymes also cut open the bacterial plasmids which have the fragments of DNA inserted into them. CfE Higher Biology Metabolism and Survival

14. Recombinant DNA tools There are over 800 endonucleases isolated from different bacteria. e.g. EcoR1 is isolated from the bacterium E.coli RY13. Each restriction enzyme recognises a short sequence of DNA bases. This is called a restriction site. The longer this sequence is the less frequently it will be found. CfE Higher Biology Metabolism and Survival

15. Recombinant DNA tools CfE Higher Biology Metabolism and Survival Restriction enzyme examples and restriction sites.

16. DNA double strand showing EcoR1 site 5’G A A T T C 3’ 3’G T T A A G5’ Mark on how EcoR1 cuts Site that has been cut showing the ends separated 5’G A A T T C 3’ 3’G T T A A G 5’ What is meant by sticky ends? The section of free nucleotides acts a sticky end for a complementary sequence

17. Recombinant DNA tools The restriction or target site may only be 4-8 nucleotides in length and is found on both DNA strands. The enzyme cuts both DNA strands and can produce blunt or sticky ends. These sticky ends and blunt ends can be sealed into a bacterial plasmid using the enzyme DNA ligase. CfE Higher Biology Metabolism and Survival

18. Recombinant DNA tools The vector used must be able to carry the DNA from the genome of one organism to that of another. An effective plasmid must have the following features. CfE Higher Biology Metabolism and Survival Restriction site Marker gene e.g. resistance to ampicillin Origin of replication

19. Recombinant DNA tools Restriction site: the same endonucleases must be able cut open the restriction site as those used to cut the DNA containing required gene. The sticky ends must be complementary. Marker gene: determines whether host cell has successfully taken up plasmid vector. e.g. if plasmid containing marker for ampicillin resistance is placed in host and then cultured in media containing ampicillin, any host cells that have not taken up the recombinant plasmid will die. They do not contain the resistance gene. CfE Higher Biology Metabolism and Survival

20. Recombinant DNA tools Origin of replication: genes that control self replication of the plasmid DNA and regulatory sequences that control existing genes and expression of inserted gene. Essential for generation of many copies of the plasmid and required gene within the bacterial host cell. If many more copies of the gene are expressed then more product can be made by fewer cells. CfE Higher Biology Metabolism and Survival

21. Recombinant DNA tools Artificial chromosomes: these have been created by scientists to act as vectors. They possess all the features of a vector but are able to carry more foreign DNA than a plasmid. A much longer sequence of DNA can therefore be carried from one organism to another. CfE Higher Biology Metabolism and Survival

22. Recombinant DNA tools Example of a commercial plasmid. Why is it useful that are there a variety of restriction sites? Different sticky ends can be chosen. CfE Higher Biology Metabolism and Survival

23. Limitations of Prokaryotes Due to the differences between eukaryotic and prokaryotic DNA, problems can arise when attempting to insert a gene from eukaryotes into prokaryotes. What are the differences between eukaryotic and prokaryotic DNA? See unit 1 notes CfE Higher Biology Metabolism and Survival

24. Limitations of Prokaryotes Another difference is that eukaryotic DNA contains introns interspersed amongst exons. What are introns and exons? Introns are non-coding regions and exons are coding regions of DNA. How are primary transcripts of mRNA modified? Introns removed from the mRNA and exons are spliced together. What is post-translational modification? Further modification to a protein which enables it to perform its specific function; may be cleavage or molecular addition. CfE Higher Biology Metabolism and Survival

25. Limitations of Prokaryotes Bacterial DNA has exons and no introns. No modification of mRNA transcripts is therefore necessary. Proteins made by bacteria do not undergo post translational modification. This means that a eukaryotic gene expressed by a prokaryotic organism may produce a polypeptide molecule which is inactive because is will not be folded correctly or will not have a post translational modification that is essential to its function. Chemicals can sometimes overcome these problems or a eukaryotic cell can be used (e.g.yeast) CfE Higher Biology Metabolism and Survival