Genetic Engineering Lab

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Genetic Engineering Lab

• Bio 101A
• April 10, 2008

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• Describe your results from the PCR lab. Was your
  sample GMO? How do you know?
• Describe differences between prokaryotes and
  eukaryotes.

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Brief Overview of Lab Objectives

1. Obtain Bacterial DNA (plasmids-pAMP and pKAN)
2. Cut DNA into specific pieces using special
   enzymes (restriction enzymes- BamHI HindIII)
3. Measure size of pieces cut by enzymes (gel
   electrophoresis)
4. Glue pieces together using other enzymes (DNA
   ligase)
5. Take glued pieces and put them into another
   bacterium (plasmid transformation of E. coli)
6. Separate bacteria with plasmid from those
   without (antibiotic selection)

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Todays Objectives

• Obtain Bacterial DNA (plasmids-pAMP and pKAN)
• Cut DNA into specific pieces using special
  enzymes (restriction enzymes- BamHI HindIII)

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Schedule

• 9am- 910 Book check
• 910-915 Review questions
• 915-935 Introduction to lab
• 935-10am Set up restriction digest/cleanup
• 10am-11am restriction digest
• 10am-11am Chi square discussion/practice
• 11am- refrigerate samples

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Lab Concepts in Detail
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Two Types of DNA in E. coli

• Chromosomal DNA necessary for cell survival
  circular, double-stranded
• Plasmid DNA extrachromosomal DNA (bonus
  material) useful for experimental manipulation
  circular, double-stranded

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Plasmids contain nonessential (but important)
genes
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ß-lactamase can destroy penicillin and other
ß-lactam antibiotics
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Kanamycin interferes with Ribosomes

• 30S ribosomal subunit is affected
• Causes frameshift in translation
• Toxic to humans

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Plasmids can be cut with restriction enzymes

• Enzymes homodimerize to make symmetrical cuts

BamHI
GATCCA GT
CG GCCTAG
sticky ends
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Restriction Enzymes cut very specific sequences
of DNA
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Plasmid DNA manipula-tion is at the heart of
biotech-nology
Cell containing gene of interest
Bacterium
Gene inserted into plasmid
Bacterial chromosome
Plasmid
Gene of interest
Recombinant DNA (plasmid)
DNA of chromosome
Plasmid put into bacterial cell
Recombinant bacterium
Host cell grown in culture to form a clone of
cells containing the cloned gene of interest
Protein expressed by gene of interest
Gene of interest
Copies of gene
Protein harvested
Basic research and various applications
Basic research on gene
Basic research on protein
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic
waste
Protein dissolves blood clots in heart attack
therapy
Human growth hor- mone treats stunted growth
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? Phage is a temperate bacteriophage

• Infects E. coli
• Genome is 46,000bp long
• dsDNA
• Sequence is known
• HindIII-digested genome is used as a molecular
  marker (ladder)

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? Phage digest is a common marker

• HindIII digest of phage genome always yields the
  same bands

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• Draw pictures of what you expect in the microfuge
  tubes from last week. Include as much detail as
  possible. What did the plasmids look like? What
  do they look like now? What else is in the tube?

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Objective(s) of the lab

• 1. Digest pAMP and pKAN with BamHI and HindIII
  restriction enzymes
• 2. Determine size of plasmids using
  electrophoresis
• 3. Create double antibiotic resistant plasmid
  using DNA ligase
• 4. Transform E. coli with new plasmid
• 5. Select for transformants using antibiotic
  media plates

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Todays Objectives

1. Ligate pAMP fragment to pKAN fragment
2. Determine fragment sizes using electrophoresis
   with HindIII ? phage digest

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Schedule

• 810-820 Lecture spiel
• 820-855 Denature/Pour gel
• 9-1015 Set up/Run gel
• 930-1015 Discuss last quiz/Drosophila/Chi-square
  
• 1015-1030 Visualize gel
• 1030-1050 Create semilog graphs of
  digest/determine fragment sizes
• 1050-11 clean up

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BamHI
BamHI
kanR
pKAN
pAMP
HindIII
ampR
HindIII
Ori
Ori
Restriction digest
Ori
BamHI
Ori
ampR
BamHI
HindIII
HindIII
kanR
BamHI
HindIII
Ligation
)
BamHI
kanR
HindIII
ampR
Ori
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T4 is a Lytic bacteriophage

• Why might a lytic bacteriophage need DNA ligase?

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Undigested plasmids are often supercoiled

• Supercoiling- increased or decreased number of
  twists/bp
• Can be caused by topoisomerases (type I and type
  II)

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Topoisomerases can cut DNA once or twice

• Either way can increase or decrease supercoiling
• Dimers can be made or removed by topoisomerases

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Supercoiled, relaxed and linear DNA do not run
equally

• Why is supercoiled faster than linear?
• Why are dimers slower than monomers?

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Week 3 Transforming Bacteria

• Review Questions
• What is our objective for the lab?
• What was accomplished for this task last week?
• How did what was done last week further our
  objectives for the lab?
• Define the following
• Plasmid
• Ligase
• Restriction Enzyme

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Week 3 Transforming bacteria

• Outline for today
• 15 min. introduction
• Transformation protocol (45 min.)
• Incubation (60 min)
• During incubation- Outline of selection (20 min)
  completion of worksheet (due Wed. noon)
• Plating- 30 min.
• Predict plating results- (10 min)

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Bacterial Transformation

• We will use chemically competent E. coli cells
• CaCl2, ice incubation, and heat shock facilitate
  the process

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Procedure

• Add 200 uL of competent bacteria to LIG vial
• Add 200 uL bacteria to any controls
• Gently mix
• Incubate on ice for 20 min

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Procedure, cont.

• Heat shock for 90 sec.
• Place back in ice for min. 60 sec.
• Add 800uL sterile LB to tube
• Incubate on shaker for 60 min.

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Sterile technique reminder

• Bacteria are ubiquitous
• Flame kills bacteria
• Any contaminants will compete with our bacteria
  of interest

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Micropipettors Review
Are fragile Expensive Precise They depend on
correct usage for accuracy
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Competent cells

• Transformation rate in normal cells is low
• Transformation rate in competent cells is higher
• We use CaCl2 to make cells chemically competent

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How can we determine if our transformation was a
success?
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Selecting for transformants

• Antibiotic-infused agar media permits only
  resistant bacteria to grow
• Our plasmids confer specific antibiotic resistance