Investigating Gene Regulation using pGLO Plasmid

1
Investigating Gene Regulation using pGLO Plasmid

• Nadja Anderson, Ph.D.
• Laurie Cale

2
Bacterial Transformation
pGLO PLasmid
Bioluminescent Bacteria
http//www.accessexcellence.org/RC/VL/GG/plasmid.h
tml
3
Students Transform Their Bacteria
4
Set up
LB AGAR AMP
LB AGAR AMP
LB Agar
5
Results
LB Agar
LB Agar Amp
LB Agar amp
No Glow ?? No Glow ??
Glow
6
Glow in the Dark Bacteria
7
Classroom PCR ???

• Isolate DNA- See Handout
• Into PCR Tube x2 each group
• 5 ml DNA
• 10 ml master mix (see handout)
• 5 ml of each primer (forward/reverse)

8
Electrophoresis
9
Electrophoresis
Primer
GFP Gene
Marker
10
Gene Expression DNA ? RNA? Protein ? Trait

• Arabinose Operon 3

Promoter
araA
AraD
araB
AraC blocks RNA polymerase binding site on
promoter
araC
Arabinose induces shape change in araC Frees RNA
polymerase binding site. araB, araA, and araD
transcriibed
11
Gene Expression DNA ? RNA? Protein ? Trait

• pGLO Plasmid

Promoter
ampR
GFP
araC
With Arabinose GFP Expression Without
Arabinose No GFP Expression
12
pGLO PLasmid
Promoter
AmpR
Source BioRad Labratories
13
Primer Design GeneBank Info

• DEFINITION Cloning vector pBAD-GFPuv, complete
  sequence (URL http//www.ncbi.nlm.nih.gov/entrez/v
  iewer.fcgi?dbnucleotideval1490531).
• ACCESSION U62637
• gene 1342.-.2061/ gene gfpuv
• 1081 accccgctta ttaaaagcat tctgtaacaa agcgggacca
  aagccatgac aaaaacgcgt
  Forward Primer
• 1342.2061 GFP Gene
• 2341 gtgcaaagac tgggcctttc gttttatctg ttgtttgtcg
  gtgaacgctc tcctgagtag
  Reverse Primer