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Key Area 2.7

Key Area 2.7. Genetic control of metabolism Growth media and environmental factors Phases of growth Control of metabolism. Word Splat!. Secondary metabolite. Growth medium. aseptic. Growth. Log. Inducer. Fermenter. Lag. death. Semi-logarithmic. stationary.

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Key Area 2.7

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  1. Key Area 2.7 Genetic control of metabolism • Growth media and environmental factors • Phases of growth • Control of metabolism

  2. Word Splat! Secondary metabolite Growth medium aseptic Growth Log Inducer Fermenter Lag death Semi-logarithmic stationary

  3. Improving Micro-Organisms Learning Intentions • Describe the methods by which wild strains of microbes can be improved for human use.

  4. Wild strains • Wild strains of micro-organisms that occur naturally can be selected and cultured • We look for desired traits, we can then improve the strain for our own benefit

  5. Strain improvement • Wild strains may have some desirable characteristics but may lack other important features: • Genetic stability • Ability to mass produce desired compounds • Strains of micro-organism can be improved by: • Mutagenesis • Selective breeding • Recombinant DNA technology.

  6. 1. Mutagenesis • Mutagenesis is the creation of mutations • The rate of mutagenesis can be increased by exposing an organism to mutagenic agents • What things do you know of that increase the risk of cancer in humans?

  7. Mutagenic agents • Mutagenic agents alter DNA and induce mutations • UV light • X-rays • Nuclear radiation

  8. Mutagenesis in microbes • Mutagenic agents are used by us to induce the production of mutant alleles in microbes • Most commonly UV light is used to mutate microorganisms which MAY produce an improved strain • Mutated strains are usually very unstable and will revert back to their original ‘wild-type’

  9. 2.Selective breeding • In organisms that reproduce sexually (fungi) new genotypes are likely to be expressed in offspring. • This can be used by scientists who will select parents with different desirable characteristics in the hope that both characteristics will be expressed in the offspring • It doesn’t always work!

  10. Super bacteria • Scientists try to create new bacteria by culturing strains of bacteria next to each other and allowing horizontal gene transfer to occur. • Can you remember the three different ways that bacteria can uptake new genetic information via horizontal transfer?

  11. Transformation • Bacterial cell takes up foreign DNA from a cell that had been destroyed

  12. Transduction • Bacterial DNA is packaged in a bacteriophage virus instead of viral DNA • Phage infects new bacteria, which incorporates the DNA as its own

  13. Conjugation • A conjugation tube (pilus) can form between 2 bacterial cells with different genetic information • They can pass and recombine their genetic information

  14. Using the information in the diagrams, summarise Transformation, transduction and conjugation.

  15. Starter Unscramble the keywords to todays lesson and match them to their definitions

  16. Starter Unscramble the keywords to todays lesson and match them to their definitions

  17. Recombinant DNA technology Learning Intentions • What is the effect of recombinant DNA technology on growth of microorganisms and their products? • How is gene expression controlled in recombinant plasmids and artificial chromosomes?

  18. Strain improvement • RDT can be used to give microorganisms genes that: • Amplify metabolic steps or remove inhibitory controls –increasing yield • Cause cells to secrete their products into surrounding medium for easier extraction • Prevents the organism surviving in an external environment - safety

  19. Artificial transformation • Artificial transformation using RDT is more commonly known as genetic engineering! • Write down what you remember about genetic engineering from N5 Biology or what you know from the news/media.

  20. Artificial transformation • Artificial transformation is is the transfer of genes from one species of organism to another enabling the second organism to produce a specific protein • E.g. an enzyme or hormone

  21. Simple steps • Gene for a desirable characteristic is identified • DNA is spliced into the DNA of a vector • Vector inserted into a host cell What does that even mean?!!!!!

  22. Vectors • Plasmids and artificial chromosomes are used as ‘vectors’ to carry information from the genome of one organism to another • Vectors transform the organism they are transferred to

  23. Restriction endonuclease • Restriction endonuclease is an enzyme that can cut up DNA and open bacterial plasmids at a restriction site. Endonuclease cuts plasmid open Endonuclease cuts out required gene DNA fragment with required genes. ‘sticky end’ at each side of gene. Plasmid (vector) with ‘sticky ends’

  24. Ligase • Ligase seals the sticky ends of the required DNA fragment to the sticky ends in the vector (plasmid) DNA fragment sealed into plasmid using ligase.

  25. Transformation • Plasmids are then inserted into bacterial cells lacking plasmids

  26. Marker Genes • Marker genes are needed to see which cells have been successfully transformed. • These are usually antibiotic resistance genes

  27. In the example above, a gene that gives the cell resistance to tetracycline is the selective marker. • Screenable markers such as the GFP gene can also be used to detect transformed cells.

  28. Health and safety • As a safety mechanism, genes are often introduced that prevent the survival of the microorganism in an external environment.

  29. Origin of replication • Genes that control self-replication of plasmid DNA and regulatory sequences • Allow control of existing gene and expression of inserted genes • Many copies of gene are expressed and more product is made by fewer cells

  30. Artificial chromosomes • Have been created by scientists to carry longer sequences of DNA into bacteria • Suggest a benefit of these chromosomes? • More genes can be transferred to the bacteria for production

  31. Limitations • Prokaryotic cells don’t carry out post or pre translational modification. No splicing occurs (Prokaryotic DNA does not contain introns) • Proteins made may be inactive because the bacteria cannot carry out the modifications • Yeast is often used instead of bacteria to overcome this issue.

  32. Ethics, risks, hazards • Pharmaceutical companies need to turn profit, less likely to research diseases that are rare or common in poor countries.

  33. New strains of micro-organism produced as a ‘result of human research’ can be patented. • Gives the scientist the sole right to make and sell product. • Hinders research and scientific progress

  34. Research • Research the development of a microbiological product from discovery to market.

  35. Tasks • Complete the questions on BST

  36. STARTER –Find someone who can answer 1 question only. Miss M Burns

  37. pGlo Transformation Learning Intentions • Carry out a practical to transform E-coli bacteria with the pGlo plasmid. • Assess and minimise risk • Interpret the findings of the investigation.

  38. Aequorea victoria

  39. DNA RNA Protein Trait Central Framework of Molecular Biology

  40. Transformation Procedures Lesson 1 Next week

  41. What is transformation? • Uptake of foreign DNA, often a circular plasmid GFP Amp Resistance

  42. ori bla What is a plasmid? • A circular piece of autonomously replicating DNA • Originally evolved by bacteria • May express antibiotic resistance gene or be modified to express proteins of interest

  43. araC ori pGLO GFP bla The many faces of plasmids Transmission electron micrograph Agarose Gel Graphic

  44. araC ori pGLO GFP bla pGLO Plasmid • Beta Lactamase • Ampicillin resistance • Green Fluorescent Protein • Aequorea victoria jellyfish gene • araC regulator protein • Regulates GFP transcription

  45. Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids Bacterial Transformation

  46. Transformation Procedure • Suspend bacterial colonies in Transformation Solution • Add pGLO plasmid DNA • Place tubes on ice • Heat shock at 42oC and place on ice • Incubate withnutrient broth • Streak plates

  47. Predictions The bacteria will not grow as it does not contain the plasmid with the ampicillin resistance gene so it will be killed of by the antibiotic. The bacteria will grow as it contains the plasmid with ampicillin resistance. The bacteria will not glow as the inducer (arabinose) is not present LB/AMP +pGlo LB/AMP -pGlo The bacteria will grow as it contains the plasmid with ampicillin resistance. The bacteria will glow as the inducer (arabinose) is present Bacteria growing as no antibiotic to kill them off LB/AMP/ARA +pGlo LB -pGlo

  48. Volume Measurement

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