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Bio 112 Genetics Lab

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					Bio 112 Genetics Lab

      Exercise 17
                        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 alleles                 Recessive alleles
 R = Purple                      r = Yellow
 SU = Starchy                    su = Shrunken

The cross which produced the corn ear above was:
                        Rr SUsu X Rr SUsu
DNA Extraction from Bacteria
Transformation of Bacteria
         (pGLO)
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
What is Transformation?




Uptake of DNA (in this case a plasmid pGLO) from the
surrounding environment of the cell.
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?
Transformation Procedure … in a
nutshell.
• Suspend bacterial colonies in Transformation Solution,
  CaCl2
• Add pGLO plasmid DNA to +DNA tube

• Place tubes on ice

• Heat shock at 42oC and place on ice

• Incubate with LB broth

• Streak plates
Expected Results
                                                       araC
 Our genes of interest:                 ori
                                                    pGLO
 amp…araC…GFP
                                                           GFP
                                              bla


 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
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.
                   araC

ori
            pGLO

                      GFP
      bla
Expected Results

				
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