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Bio 112 Genetics Lab
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  1. Bio 112 Genetics Lab Exercise 17

  2. Dihybrid Cross in Corn There are four grain phenotypes in the above ear of corn: Purple & Starchy(A), Purple & Sweet(B), Yellow & Starchy(C) and Yellow & Sweet(D). These four grain phenotypes are produced by the following two pairs of heterozygous genes (R & r and SU & su) located on two pairs of homologous chromosomes (each gene on a separate chromosome): Dominant allelesRecessive alleles R = Purple r = Yellow SU = Starchy su = Shrunken The cross which produced the corn ear above was: RrSUsu X RrSUsu

  3. DNA Extraction from Bacteria

  4. Transformation of Bacteria (pGLO)

  5. We will introduce a gene into E. coli that will produce a protein (green fluorescent protein-GFP) that will cause the colonies to glow green when exposed to ultraviolet light

  6. What is Transformation? Uptake of DNA (in this case a plasmid pGLO) from the surrounding environment of the cell.

  7. What is Green Fluorescent Protein-GFP)? GFP was discovered in the bioluminescent jelly Aequorea victoria. The gene that makes this protein is used extensively in research…and also for fun?

  8. Transformation Procedure … in a nutshell. • Suspend bacterial colonies in Transformation Solution, CaCl2 • Add pGLO plasmid DNAto +DNA tube • Place tubes on ice • Heat shock at 42oC and place on ice • Incubate withLB broth • Streak plates

  9. Expected Results

  10. araC ori pGLO GFP bla Our genes of interest: amp…araC…GFP • amp – this gene will give our transgenic bacteria resistance to the antibiotic ampicillin • araC – this gene will produce a protein which in the presence of the sugar arabinose will allow the bacteria to turn on the GFP gene • GFP – in the presence of arabinose, this gene will “turn on” and cause the transformed (transgenic) bacteria to glow green

  11. The Role of Arabinose • The bacterial genes that make the digestive enzymes needed to break down arabinose for food are not expressed (made) when arabinose is absent. • When arabinose is present the genes are turned “on”. When it is absent the genes remain “off”. • Arabinose initiates transcription of the genes by promoting the binding of RNA polymerase to the promoter.

  12. araC ori pGLO GFP bla

  13. Expected Results