Genetic Engineering Lab Bio 101A. Brief Overview of Lab Objectives 1.Obtain Bacterial DNA (plasmids-pAMP and pKAN) 2.Cut DNA into specific pieces using.

Genetic Engineering Lab

Bio 101A

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)

Today’s Objectives

1. Obtain Bacterial DNA (plasmids-pAMP and pKAN)

2. Cut DNA into specific pieces using special enzymes (restriction enzymes- BamHI; HindIII)

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

Lab Concepts in Detail

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

Plasmids contain nonessential (but important) genes

β-lactamase can destroy penicillin and other β-lactam antibiotics

Kanamycin interferes with Ribosomes

• 30S ribosomal subunit is affected

• Causes frameshift in translation

• Toxic to humans

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

Restriction Enzymes cut very specific sequences of DNA

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

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)

λ Phage digest is a common marker

• HindIII digest of phage genome always yields the same bands

• 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?

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

Today’s Objectives

1. Ligate pAMP fragment to pKAN fragment2. Determine fragment sizes using

electrophoresis with HindIII λ phage digest

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

pAMP pKAN

ampR

BamHI

HindIII

OriHindIII

BamHI

Ori

kanR

Restriction digestBamHI

HindIII

BamHI

HindIII

OriOri

BamHIHindIII

ampR

kanR

ampR

kanRBamHI

HindIII

Ori

Ligation )

T4 is a Lytic bacteriophage- the source of our T4 DNA ligase

• Why might a lytic bacteriophage need DNA ligase?

Undigested plasmids are often supercoiled

• Supercoiling- increased or decreased number of twists/bp

• Can be caused by topoisomerases (type I and type II)

Supercoiled, relaxed and linear DNA do not run equally

• Why is supercoiled faster than linear?

• Why are dimers slower than monomers?

Topoisomerases can cut DNA once or twice

• Either way can increase or decrease supercoiling

• Dimers can be made or removed by topoisomerases

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

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)

Bacterial Transformation

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

process

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

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.

Sterile technique reminder

• Bacteria are ubiquitous• Flame kills bacteria• Any contaminants will

compete with our bacteria of interest

Micropipettors Review

Are fragile Expensive PreciseThey depend on correct usage for accuracy

Competent cells

• Transformation rate in normal cells is low

• Transformation rate in competent cells is higher

• We use CaCl2 to make cells chemically competent

How can we determine if our transformation was a success?

Selecting for transformants

• Antibiotic-infused agar media permits only resistant bacteria to grow

• Our plasmids confer specific antibiotic resistance

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

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

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