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 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 : Review questions : Introduction to lab am: 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. coli Chromosomal 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 enzymes Enzymes homodimerize to make symmetrical cuts CG GCCTAG GATCCA GT “sticky ends” C G G A T C C A G 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 Bacterial chromosome Plasmid Gene inserted into plasmid Cell containing gene of interest Gene of interest DNA of chromosome Recombinant DNA (plasmid) 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 Protein harvested Gene of interest Copies of gene Basic research on gene Basic research on protein Basic research and various applications 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

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 fragment 2.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 amp R BamHI HindIII Ori HindIII BamHI Ori kan R Restriction digest BamHI HindIII BamHI HindIII Ori BamHI HindIII amp R kan R amp R kan R BamHI 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 Bacteria Review 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.Plasmid b.Ligase c.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 fragileExpensivePrecise They 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 results GroupLB onlyLB-ampLB-KanLB-Amp/Kan (-) control (+) control pAMPLawnNG (+) control pKANLAWNNG Sarai, Zina, Lynette, Raymond LawnNG Steph, Kwan, Heather, June lawnNG Yellow-green mold (lawn) Jessica, Minh, TiffanyBig lawn1 mold colony3 coloniesNG Jake, YadanarLawnNG 1 dark green colongy Contamination Alena, Tim, JuliaBig lawn2 large mold colonies 1 yellow/1 clear NG Juneto, AnnieLawnNG Max, Xiaozhu, KenisLawnNG Tony, Mengting, Xaviera LawnNG

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