genetic engineering lab bio 101a. brief overview of lab objectives 1.obtain bacterial dna...
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Genetic Engineering Lab
Bio 101A
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Brief Overview of Lab Objectives1. 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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Today’s Objectives
1. Obtain Bacterial DNA (plasmids-pAMP and pKAN)
2. 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. coliChromosomal 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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β-lactamase can destroy penicillin and other β-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 enzymesEnzymes homodimerize to make symmetrical cuts
CGGCCTAG
GATCCAGT
“sticky ends”
C G G A T C C AG C C T A G G T
BamHI
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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
Bacterium
Bacterialchromosome
Plasmid
Gene inserted intoplasmid
Cell containing geneof interest
Gene ofinterest DNA of
chromosome
RecombinantDNA (plasmid)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest
Protein expressedby gene of interest
Protein harvested
Gene ofinterest
Copies of gene
Basicresearchon gene
Basicresearchon protein
Basic research andvarious applications
Gene for pestresistance insertedinto plants
Gene used to alterbacteria for cleaningup toxic waste
Protein dissolvesblood clots in heartattack therapy
Human growth hor-mone treats stuntedgrowth
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λ 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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λ Phage digest is a common marker
• HindIII digest of phage genome always yields the same bands
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• On scratch paper, or in an unimportant region of your GE section of your lab notebook:
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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Today’s Objectives
1. Ligate pAMP fragment to pKAN fragment2. Determine fragment sizes using
electrophoresis with HindIII λ phage digest
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Schedule• 8:10-8:20 Lecture spiel• 8:20-8:55 Denature/Pour gel• 9-10:15 Set up/Run gel• 9:30-10:15 Discuss last quiz/Drosophila/Chi-
square• 10:15-10:30 Visualize gel• 10:30-10:50 Create semilog graphs of
digest/determine fragment sizes• 10:50-11 clean up
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pAMP pKAN
ampR
BamHI
HindIII
OriHindIII
BamHI
Ori
kanR
Restriction digestBamHI
HindIII
BamHI
HindIII
OriOri
BamHIHindIII
ampR
kanR
ampR
kanRBamHI
HindIII
Ori
Ligation )
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T4 is a Lytic bacteriophage- the source of our T4 DNA ligase
• 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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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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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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Week 3: Transforming BacteriaReview Questions
1. What is our objective for the lab?2. What was accomplished for this task last week?3. How did what was done last week further our
objectives for the lab?4. Define the following:
a. Plasmidb. Ligasec. Restriction Enzyme
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Week 3: Transforming bacteria
Outline for today• 10 min. introduction• Transformation protocol (30 min.)• Incubation (50 min)• During incubation-• Plating- 20min.• 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 PreciseThey 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
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Today’s outline
• Check GE Results• Share results as a group• Set up Yeast fermentation lab• Incubate• During incubation:– Discuss lab report– Discuss practical– Discuss iGEM presentation
• Take results of Yeast incubation lab
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Report your resultsGroup LB only LB-amp LB-Kan LB-Amp/Kan
(-) control
(+) control pAMP Lawn NG NG NG
(+) control pKAN LAWN NG NG NG
Sarai, Zina, Lynette, Raymond
Lawn NG NG NG
Steph, Kwan, Heather, June
lawn NG NG Yellow-green mold (lawn)
Jessica, Minh , Tiffany Big lawn 1 mold colony 3 colonies NG
Jake, Yadanar Lawn NG NG 1 dark green colongy Contamination
Alena, Tim, Julia Big lawn 2 large mold colonies
1 yellow/1 clear
NG
Juneto, Annie Lawn NG NG NG
Max, Xiaozhu, Kenis Lawn NG NG NG
Tony, Mengting, Xaviera
Lawn NG NG NG
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Why didn’t things go right?• Maybe we killed the bacteria when we spread it, but
we did a good job with the LB plates• Maybe the temperature for the heat shock was too
high, and we killed the bacteriaX• Maybe we heat shocked for too long• Maybe we didn’t return the bacteria to the ice quick
enough• Maybe the DNA ligase didn’t work• Maybe we didn’t denature our restriction enzymes
successfully• Maybe some guy in the stockroom didn’t pour the
plates right