Ch. 5A: Transforming Bacteria with Recombinant Plasmids
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- Slide 1
- Ch. 5A: Transforming Bacteria with Recombinant Plasmids
- Slide 2
- Learning goals Describe the role of transformation in the gene
cloning process Explain the purpose of each control in the
transformation experiment Explain how the information encoded in a
gene is expressed as a trait
- Slide 3
- Key Ideas A recombinant plasmid must be taken up by bacteria
bacteria cell machinery used to replicate and to express the gene
of interest. If transformed with the pARA-R plasmid bacteria can be
identified Ampicillin will prevent the growth of cells that do not
carry an ampicillin resistance gene Arabinose will activate the
bacteria promoter that controls expression of the rfp gene.
- Slide 4
- Go to pg 76 and complete Lab 5A Transforming Bacteria with the
pARA-R PlasmidTransforming Bacteria
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- RFP expression araC generfp geneP BAD Transcription mRNA
Translation araC protein Biotech Experience
- Slide 6
- RFP expression rfp geneP BAD araC protein araC gene araC
protein prevents RFP transcription by causing a loop to form in the
region of the fp gene r Biotech Experience
- Slide 7
- RFP expression araC protein arabinose araC generfp geneP BAD
arabinose araC protein complex RNA polymerase Arabinose araC
protein complex prevents DNA looping and helps to align RNA
polymerase on the promoter site (P BAD ). mRNA Transcription
Translation RFP (red fluorescent protein) Biotech Experience
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- Plating Tips Note the plate markings: I=LB, II=LB/amp,
III=LB/amp/ara Label the bottom of the plate near the edge Open the
plates like clam shells Sample goes on the agar, not the lid
- Slide 9
- More Plating Tips Agar is like jello, firm but not invincible,
be gentle the spreader is not a shovel Turn the plates upside down
(lids down) for incubation, stacked and taped together After
incubation, do not open plates, observe through the bottom
- Slide 10
- Remind Students 1. Sterile technique Using bacteria
Contamination may affect results 2. Carefully READ and FOLLOW the
lab protocol. Be sure lab partners communicate 3. No Food or
Drinks
- Slide 11
- Sterile technique Always follow the protocol carefully know
what youre doing Work quickly. Less time = Less opportunities for
contamination Do not leave any container (tube, plate) open any
longer than needed Watch what your equipment touches there is no 5
second rule here. All tips, tubes and spreaders go in the
contaminated waste container
- Slide 12
- DO NOT open plates, observe by viewing through the bottoms Used
plates dispose in the contaminated waste bags LB P-P+ LB/amp
LB/amp/ara P-P+
- Slide 13
- oops plates
- Slide 14
- Slide 15
- Satellite colonies Some cells without antibiotic resistance do
become "freeloaders" and survive because other cells are doing the
work of destroying the antibiotic in their immediate vicinity on
the plate. They only develop with antibiotics such as ampicillin,
that are destroyed by enzymes such as beta lactamase outside of the
cell.
- Slide 16
- Why do we get satellite colonies? The ampicillin plate is old
(meaning that the antibiotic is partially degraded) The transformed
cells are plated at very high density (meaning that the plate is
covered with huge number of cells) The copy number of the plasmid
in the cells is so high that beta lactamase is secreted at high
levels, The colonies grow on the plate for several days (allowing
more time for degradation).